Supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) (no. 262011-2009) to CV and March of Dimes Social and Behavioral Sciences Research grant (#12-FY12-179) to CV and JTS; by studentships to LSF from the Minist&#232;re de l'&#233;ducation, de l'Enseignement sup&#233;rieurs et de la recherche (MEESR)-Fonds de recherche du Qu&#233;bec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the R&#233;seau Qu&#233;b&#233;cois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Sant&#233;, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cient&#236;fico e Tecnol&#242;gico (CNPq) and the Programme de bourses d'excellence pour &#233;tudiants &#233;trangers MEESR-FRQNT.

Placentas were obtained immediately after spontaneous vaginal deliveries from uncomplicated pregnancies at the CHUM-St-Luc Hospital, Montreal, QC, Canada, with informed patient consent and approval of ethical committees (CHUM-St-Luc Hospital and INRS-Institut Armand-Frappier, Laval, QC, Canada).
1. Isolation and Purification of Villous Cytotrophoblast Cells
<ol>
	<li>Solutions and media
	<ol>
		<li>Prepare transport media by supplementing Dulbecco&#39;s Modified Eagle&#39;s Medium High-Glucose (DMEM-HG) with 1% vol/vol antibiotic (10,000 units/ml penicillin G, 100 mg/ml streptomycin sulphate) and store at 4 &#176;C.</li>
		<li>Prepare primary culture media by supplementing DMEM-HG with 10% vol/vol fetal bovine serum (FBS), 25 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), 1% vol/vol antibiotic (10,000 units/ml penicillin G, 100 mg/ml streptomycin sulphate) and store at 4 &#176;C. Warm media to 37 &#176;C before use.</li>
		<li>Prepare 4 L of saline solution (0.9% weight/vol sodium chloride).</li>
		<li>Prepare modified Hank&#39;s Balanced Salt Solution (HBSS) by adding 25 mM HEPES to 1x HBSS (pH 7.4).</li>
		<li>Prepare fresh, four bottles of digestion solution with modified HBSS (prepared in 1.1.4), magnesium sulfate (MgSO4), calcium chloride (CaCl2), trypsin, deoxyribonuclease IV (DNase IV), and 1% vol/vol antibiotic (10,000 units/ml penicillin G, 100 mg/ml streptomycin sulfate) as shown in Table 1.</li>
		<li>Prepare density centrifugation media gradient
		<ol>
			<li>Prepare density centrifugation media solution by supplementing density centrifugation media with 10% vol/vol HBSS 10x.</li>
			<li>Prepare 14 assay tubes with density centrifugation media solution and modified HBSS, as described in Table 2.</li>
			<li>Mix each density solution (step 1.1.6.2) vigorously before adding its content to the gradient. Gently add the density solutions to a 50 ml glass centrifuge tube with a peristaltic pump (1 ml/min), beginning with the highest concentration (70%). Avoid droplets by draining the solutions against the tube wall to maintain the proper separation of each layer of the gradient.
			<ol>
				<li>In absence of a peristaltic pump, apply the layers very gently with Pasteur pipettes.</li>
			</ol>
			</li>
		</ol>
		</li>
		<li>Prepare final running buffer by supplementing the running buffer (see Table of Materials) with 2% vol/vol antibiotic/antimycotic (10,000 units/ml penicillin G, 100 mg/ml streptomycin sulfate). Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Villous cytotrophoblast isolation 
	Note: Use sterile surgical equipment, glassware, pipettes, flasks, etc.
	<ol>
		<li>On the day of villous cytotrophoblast isolation, in a 37 &#176;C water bath, warm the digestions solutions (from step 1.1.5) and 70 ml of FBS. Note: Use 50 ml of FBS to interrupt the digestions and the remaining 20 ml for the freezing step&#160;(1.2.22) which can be placed on ice once thawed.</li>
		<li>After delivery, bring the placenta to the laboratory in ice-cold transport medium (from step 1.1.1) as quickly as possible (less than 1 hr).</li>
		<li>Discard transport medium and placental blood in liquid dustbin. Weigh the placenta and immerse in cold saline solution.</li>
		<li>Measure and analyze the following features: umbilical cord length; umbilical cord localization; placental length, width, shape (oval, discoid); membrane color; cotyledon structure pathologies. Note: Data are presented in the results section.</li>
		<li>Cut the umbilical cord alongside its placental insertion (i.e., at its base with an additional 1 cm radius circle around the cord). Immerse in 300 ml of histological tissue fixative solution (formalin 10%) for later histological analysis.</li>
		<li>Cut the entire placenta into cubes of 5 x 5 x 5 cm. Wash thoroughly (4 times x ~ 1 min) in saline solution (0.9%) to remove blood cells until saline solution is clear. Discard rinsing liquid.</li>
		<li>In a watch glass, remove placental membranes and mince tissues to remove blood vessels and calcifications. Hold blood vessels firmly with forceps and remove tissues using the back of Metzenbaum scissors.
		<ol>
			<li>Place minced placenta in a B&#252;chner funnel. Rinse with approximately 100 ml of saline buffer. Continue mincing until 30 - 35 g of minced tissue is obtained (use plastic weighing boat and scale). If needed, mince the remainder of the placenta to obtain up to three additional 30 - 35 g preparations. During this time, put minced tissue in a weighing boat on ice. 
			Note: This step will take around 45 min to 1 hr.</li>
		</ol>
		</li>
		<li>Add the 30 - 35 g minced placental tissue to a trypsinizing flask. Transfer 150 ml of the prepared digestion solution 1 (Table 1) to the trypsinizing flask and mix well.</li>
		<li>Place the trypsinizing flask in a shaking water bath for 30 min at a speed of no more than 50 cycles/min and manually mix the trypsinizing flask every 5 min for homogenous digestion.</li>
		<li>At the end of the first digestion, remove the trypsinizing flask from the water bath and tilt it (45&#176;) for 1 min to sediment the placental tissue. With a 10 ml sterile pipette, remove and discard approximately 80 ml of supernatant. Avoid aspirating the tissue.</li>
		<li>Transfer 100 ml of digestion solution 2 (Table 1) to the trypsinizing flask; mix well and repeat step 1.2.9.</li>
		<li>At the end of the second digestion, remove the trypsinizing flask from the water bath and tilt it 45&#176; for 1 min. With a 10 ml sterile pipette, remove 80 ml supernatant and gently transfer to a centrifuge tube with a cell strainer (100 &#181;m mesh). Transfer the filtered supernatant to a beaker containing 2 ml of FBS every time the centrifuge tube is full.</li>
		<li>Perform the third digestion as described for the second digestion (1.2.11 and 1.2.12) using 75 ml digestion solution 3. In parallel, perform steps 1.2.15 to 1.2.16.1 for digestion 2.</li>
		<li>Perform the fourth digestion exactly as the third digestion using the digestion solution 4 (75 ml), but collect a maximum amount of supernatant. Perform steps 1.2.15 to 1.2.16.1 in parallel for digestion 3, and finally for digestion 4.</li>
		<li>Aliquot the supernatant (from digestions 2, 3 and 4) into 13.5 ml parts, each part into one 15 ml centrifuge tube. With a 22.8 cm long glass Pasteur pipette, very gently and slowly place 1.5 ml of FBS at the bottom of each tube in order to create a separate layer. Do not mix FBS and supernatant. Centrifuge the tubes without brake for 20 min at 1,250 x g at room temperature. 
		Note: After centrifugation, 4 layers are visible in the tube, as shown in Figure 1. The separation of trypsin and trophoblast cells avoids excessive cellular digestion.</li>
		<li>With a vacuum pump, aspirate and discard the supernatant (digestion solution) and FBS layers, including the whitish film between the two layers. Resuspend the pellet (the trophoblasts and red blood cell layers) with 1 ml of warm cell culture medium (step 1.1.2; with no FBS and no HEPES).
		<ol>
			<li>Collect resuspended cells from all tubes and combine them in 1 tube. Let the tube stand at room temperature until the end of all digestions. Note: After all digestions, the usual yield is 3 tubes of resuspended cells (one per digestion).</li>
		</ol>
		</li>
		<li>Make up the volume to 15 ml with warm cell culture medium. Centrifuge at 1,250 x g for 10 min at room temperature. Remove the supernatant with a vacuum pump. Avoid aspirating the pellet.</li>
		<li>Gently resuspend the pellet obtained from the 3 tubes with 1 ml of warm cell culture medium. Pool their content in 1 tube. To obtain 8 ml, complete the volume with warm cell culture medium.</li>
		<li>Very gently layer the cell suspension on a separation gradient with a Pasteur pipette. Centrifuge without brake for 30 min at 507 x g at room temperature.</li>
		<li>After centrifugation, identify the different layers of cells in the gradient with back-lighting. Locate the layers containing trophoblast and contaminating cells between 40 - 50% of density centrifugation medium. With a vacuum pump, remove upper layers (&#62; 50%).</li>
		<li>Collect cells located in the layers of interest with a Pasteur pipette and transfer them to a 50 ml centrifuge tube. Make up the volume to 50 ml with cell culture medium. Centrifuge for 10 min at 1,250 x g at room temperature.</li>
		<li>Under sterile conditions, discard the supernatant, resuspend the pellet with 20 ml of FBS and count the number of cells using a hemocytometer. On ice, add 2.22 ml of sterile dimethyl sulfoxide (DMSO) and mix gently by flipping. Aliquot 1.5 ml of cell suspension into cryogenic vials, freeze overnight at -80 &#176;C and transfer to a liquid nitrogen tank.</li>
	</ol>
	</li>
	<li>Trophoblast purification
	<ol>
		<li>Install the rinsing and running buffers and a new filter column on the magnetic purification instrument according to manufacturer&#39;s instructions. Perform the &#34;clean program&#34; to clean negative 1, positive 1 and positive 2 ports, and then introduce the 50 ml tubes under each port according to manufacturer&#39;s instructions.</li>
		<li>Thaw the cells that were frozen in step 1.2.22 quickly in a 37 &#176;C water bath. Transfer cells to a 50 ml tube and resuspend cells gently with 20 ml of cold running buffer solution. Centrifuge the tube for 5 min at 450 x g and 4 &#176;C.</li>
		<li>Discard the supernatant. Repeat the wash step with cold running buffer. Count cells using a hemocytometer to determine viability. Repeat the centrifugation (for 5 min, 450 x g at 4 &#176;C). Carefully remove the supernatant. Add 1 ml of cold running buffer containing 1% vol/vol of mouse anti-HLA-ABC antibodies. Incubate at 4 &#176;C for 30 min, mixing gently every 5 min.</li>
		<li>Add 6 ml of cold running buffer. Centrifuge for 5 min at 450 x g and 4 &#176;C. Discard the supernatant and repeat this step. Resuspend cells in 1 ml of cold running buffer containing 10% vol/vol of anti-mouse secondary antibody-coupled magnetic beads. Incubate at 4 &#176;C for 15 min, mixing gently every 5 min.</li>
		<li>Add 6 ml of cold running buffer. Centrifuge for 5 min at 450 x g and 4 &#176;C. Discard the supernatant and resuspend in 5 ml cold running buffer.</li>
		<li>Separate the trophoblast cells using the magnetic purification instrument. Collect cells at the negative port and add 20 ml of cold running buffer. 
		Note: Trophoblast cells do not contain the complex HLA-ABC, and are thus separated from other cell types and directed towards the negative 1 port.</li>
		<li>Centrifuge for 5 min at 450 x g and 4 &#176;C. Discard the supernatant and gently resuspend the cells in 20 ml warm primary culture medium. Count the cells using a hemocytometer to determine viability.</li>
		<li>Plate the cells at the following densities: 0.15 x 106 cells/well in 96-well plates, 1.6 x 106 cells/well in 24-well plates and 4.5 x 106 cells/well in 6-well plates. Incubate plates at 37 &#176;C and 5% CO2.</li>
		<li>Confirm the purity of the trophoblast cells by flow cytometry16,17 and/or by immunocytochemistry18. 
		Note: The purity of the immunopurified cells was determined using FITC-conjugated monoclonal antibodies against cytokeratin-7 and vimentin17-19. This protocol is well detailed in Lanoix et al., 20087.</li>
		<li>After at least 4 hr, rinse the cells twice with warm culture medium to remove unattached cells and then transfer the plates to the normoxia chamber, which is composed of 8% O2 (see Figure 2 and section 2).</li>
	</ol>
	</li>
</ol>
2. In Vitro Induction of Normoxia and Hypoxia/Reoxygenation
<ol>
	<li>Incubator chamber operation (see Figure 2A for set-up).
	<ol>
		<li>In a laminar flow hood, place a Petri dish containing sterile water at the bottom of the incubator chamber to avoid dryness; then place the previously prepared cell culture plates or flasks (step 1.3.10) on the superior shelves of the chamber.</li>
		<li>Outside the hood, attach the chamber (inlet port) to the gas hose (Figure 2A: 5a, b and c) to reach the tube of gas (8% O2 or 0.5% O2) (Figure 2A: 7). Open both inlet and outlet ports of the chamber. At this moment, the gas regulator (Figure 2A: 4) should remain closed.</li>
		<li>Carefully open the gas regulator valve (Figure 2A: 4). Flush for 4 min with an air flow of 25 L/min to completely replace the air inside the chamber.</li>
		<li>After flushing the chamber, close the gas regulator then the inlet and outlet ports of the chamber.</li>
		<li>Unplug the flow meter outlet hose (Figure 2A: 5c) from the inlet port of the chamber and place the chamber in a cell culture incubator at 37 &#176;C.</li>
		<li>Replace the air currently present in the plates, flasks and dissolved in the culture medium by filling the chamber with gas 1 hr after step 2.1.3.</li>
		<li>Repeat steps 2.1.1 to 2.1.6 for the other gas compositions (e.g., 2% O2 for first trimester trophoblast culture9).</li>
	</ol>
	</li>
	<li>Confirm the oxygen percentage (Figure 2B-C)
	<ol>
		<li>To confirm the concentration of oxygen in the cell culture medium (without cells) inside the chamber, use an oxygen electrode connected to an oxygen adapter. Connect the oxygen electrode to a voltmeter.</li>
		<li>Create a calibration curve in the same solution (i.e., cell culture medium) by exposing the solution to gases with known oxygen contents (e.g., 0% and 21% oxygen). After the readings are stabilized for each concentration, introduce the electrode into the medium in the chamber. 
		Note: Take all measurement at the same depth in order to avoid any bias in oxygen concentration10.</li>
	</ol>
	</li>
	<li>Induction of normoxia and hypoxia/reoxygenation in trophoblasts.
	<ol>
		<li>After adding primary trophoblast cells to the appropriate cell culture flasks, plates or Petri dishes, perform treatments as necessary.</li>
		<li>In parallel, inside the chamber, expose cells to the desired gas mixture to reproduce a specific condition every 24 hr (Figure 3).</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

In mammals, fetal development is directly dependent on adequate placental function. The developmental origins of health disorders are based on the hypothesis that the cause of diseases manifested later in life can be traced back to early development and that the placenta has a mechanistic role in fetal programming30-32. The placenta is the key mediator of fetal growth and development: it regulates nutrient transfer, protects against harmful exposures, and has major endocrine functions. The development by Kliman et al. of a reproducible technique to isolate viable primary cytotrophoblasts is a milestone in the study of normal and abnormal placental functions6. Many researchers have adapted this technique to reproduce specific conditions in vitro to understand placental physiology18,20,33,34. As described by Petroff et al., many steps are important to guarantee a purified and robust yield of isolated cytotrophoblast cells18. For example, the digestion steps have undergone several modifications since the development of the technique in 1988, such as an increased number and length of the digestions, as well as to the composition and quantity of digestion enzymes, resulting in greater numbers of viable isolated cytotrophoblasts18. This current protocol has three main advantages: cryopreservation, which allows for the possibility to continue the protocol later immunomagnetic purification, which increases cytotrophoblast purity and the use of pre-coated microplates improving cell attachment7,34-36. The existing literature contains several examples of diverse modifications of the isolation technique for cytotrophoblast cells, but the characteristics of the density centrifugation media has remained virtually unmodified37. The presented technique of isolation/immunopurification has several critical steps. Hence, it is important to evaluate the purity of the villous cytotrophoblast cells at the end of each immunopurification. This can be done by flow cytometry using appropriate antibodies as markers: cytokeratin-7 (trophoblastic marker), cluster of differentiation 45 (CD45) and vimentin (non-trophoblast cells markers)18,38,39. Other critical aspects are the quality of the obtained placentas, the FBS and density centrifugation media gradient, as well as centrifugation speed, which can all influence the yield and quality of the cells20,40.
Although widely used, the present technique has unavoidable limitations. Firstly, the amount of viable cytotrophoblasts cells obtained after immunopurification is relatively low and is the main limiting factor in the number of possible conditions/treatments that can be tested. Secondly, the life-span of primary trophoblast cells is short and in vitro differentiation into syncytiotrophoblast is closely followed by a reduction of cell viability and an increase of apoptosis. The short length of trophoblast cell viability which does not proliferate in vitro, limits the evaluation of longer term treatments, because apoptosis is irreversibly triggered after about 4 days of culture7,41. Thirdly, interplacental variability is large, so a relatively large number of placentas is required to obtain statistically interpretable results. On the other hand, primary trophoblast culture has unique advantages, such as the capacity of the cells to differentiate into syncytium, which allows for the study of conditions and treatments in different cell phenotypes according to the various stages of differentiation. The oxygenation method presented in this protocol is highly adaptable and its configuration can be tailored for other situations, such as the culture of primary trophoblast cells from first trimester pregnancy, which should be exposed to a lower oxygen tension for normoxia9,42,43.
There are other approaches to study human placental function in vitro. Snap-freezing placental tissues allows for multi-omics analyses, but requires placental multisite sampling, to avoid for example metabolic variations due to the oxygenation gradient, which decreases from the central villi to the periphery44. However, live trophoblast cell biology and behavior cannot be studied using this approach45. Villous explants have the advantage of maintaining the whole villous structure with the constituent cell types and their communication, but responses to treatments are not specific to trophoblast cells23. Commercially available trophoblast-like choriocarcinoma cell lines, such as BeWo, Jeg-3 and JAR can be used to study placental functions, such as fusion, differentiation, and transplacental transport. However, recent studies show that gene expression in primary cytotrophoblasts and BeWo tumor cells are poorly correlated46-48. Thus, primary villous trophoblast cell culture, despite its limitations, possess the unique advantage of mimicking the in vivo environment of the normal or abnormal placenta.
Studies using villous trophoblast cell in primary culture and villous explants show that hypoxia and hypoxia/reoxygenation systematically decrease trophoblast cell viability, concomitant with increased levels of oxidative stress, inflammation, autophagy and apoptosis43,49-52. This hypoxia/reoxygenation cell culture model, specifically, has allowed us to demonstrate the antioxidant and anti-apoptotic effects of melatonin in villous trophoblast cells. In primary villous trophoblast cells exposed to hypoxia/reoxygenation, melatonin prevents the following: induction of oxidative stress, decreased antioxidant enzyme activities, increased activity of redox-sensitive signaling pathways, and induction of mitochondrial apoptosis (Figure 5B)8. The hypoxia/reoxygenation model is a unique tool to ascertain the preventive role of melatonin in oxidative stress-induced damage and its possible protective role in pregnancy complications such as preeclampsia, where placental melatonin synthesis is reduced53. Melatonin is a powerful antioxidant with a wide range of targets54. Also, the safety of melatonin as a treatment has been largely established. The beneficial results with melatonin have been reproduced by several researchers and melatonin is currently in clinical trials as a potential preventive or therapeutic treatment in pregnancies complicated by preeclampsia or intrauterine growth restriction55,56.
In conclusion, the isolation, purification and primary culture of high quality cytotrophoblast cells, together with the technique of hypoxia/reoxygenation enable a wide range of promising experimental approaches to better understand pregnancy complications related to oxidative stress and improve placental health.

Throughout human pregnancy, the placental cytotrophoblast cells, which are mononucleated stem cells, rapidly proliferate and differentiate into either villous or extravillous cytotrophoblast cells. Extravillous cytotrophoblasts invade and remodel the spiral arteries of the uterine wall. Villous cytotrophoblasts, on the other hand, continue to proliferate, differentiate and fuse to form multinucleated syncytiotrophoblast (the syncytium)1. The maintenance of villous trophoblast homeostasis is essential for fetal well-being and healthy pregnancy. In fact, villous trophoblasts allow maternal-fetal exchange of oxygen and nutrients, and produce essential hormones for pregnancy. Moreover, the syncytiotrophoblast is the only cell-type in direct contact with the maternal blood circulation and provides an essential physical and immunological barrier. Therefore, the syncytiotrophoblast must undergo apoptosis and replacement for homeostatic maintenance and to avoid placental pathologies2-5.
The technique developed by Kliman et al.6 in 1986 to isolate primary villous cytotrophoblasts from human placentas caused a revolution in placental research by allowing the study of the molecular mechanisms involved in villous trophoblast differentiation. This classical technique, based on sequential enzymatic digestions with trypsin and DNase, followed by isolation in density centrifugation media (colloidal silica particles coated by polyvinylpyrrolidone, or Percoll) is now recognized as the gold standard for isolating villous cytotrophoblast cells. The technique can be optimized by magnetic immunopurification, a procedure that separates villous cytotrophoblasts from non-trophoblastic cells based on the differential expression of specific antigens on the surfaces of these cells. We chose the human leukocyte antigen ABC (HLA-ABC) due to the absence of its expression on the trophoblastic cell membrane7,8.
The placenta is an organ that undergoes dramatic variations in oxygen levels during pregnancy. In the first trimester, the oxygenation ratio is physiologically very low (2% O2) but increases to mild levels of oxygenation (8% O2) in the second and third trimester. Tuuli et al.9 described that the in vitro reproduction of the trophoblast environment inside the placental villi is a challenge and variations in oxygenation levels may even lead to phenotypical changes. It is, therefore, suggested to adopt 8% oxygen as normoxia to mimic the oxygen tension found in placental villi during the third trimester of gestation8,9. Chen et al.10 extensively studied several variables related to oxygen tension in trophoblast cell culture and demonstrated the importance of determining oxygen levels in a pericellular environment. The levels of oxygen in the villi tend to increase due to vasculogenesis. The blood flow in placental villi increases constantly and the level of hydrogen peroxide (an abundant reactive oxygen species) is an important signal that controls vasculogenesis11,12. In pregnancy complications, a lack of vasculogenesis generates hypoxia, and more importantly, intermittent variations of oxygenation (called hypoxia/reoxygenation). These conditions lead to an abnormal increase in oxidative stress, which compromises placental and fetal viability13,14. The alterations that trophoblast cells undergo in vivo during episodes of hypoxia/reoxygenation can be mimicked in vitro as follows: villous cytotrophoblasts are maintained under normoxic conditions (8% O2) until they differentiate into syncytiotrophoblast. They are then subjected to hypoxic conditions (0.5% O2) for 4 hr, followed by an additional 18 hr of normoxia (reoxygenation). Using this hypoxia/reoxygenation approach, trophoblasts exhibit deregulated redox status and increased levels of intrinsic apoptosis8, as has been observed in certain pregnancy complications. Hence, this is a useful in vitro model to evaluate new preventive and therapeutic approaches to combat pregnancy complications associated with placental hypoxia/reoxygenation.
Placental cells produce melatonin, which has several important functions, such as an ability to obviate oxidative stress and placental dysfunction15. Here, we present the experimental approach and cell models used to demonstrate the protective effects of melatonin in placental trophoblast cells at the molecular, cellular and functional level8.

<table><tbody><tr><td>Curved Metzenbaum Scissors</td><td>Shandon</td><td>9212</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Splinter Forceps Fine 41/2 in</td><td>Fisherbrand</td><td>13-812-42</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Scissors 4.5 Str Dissection</td><td>Fisherbrand</td><td>08-940</td><td>surgical equipment (cell isolation) (2 units)</td></tr><tr><td>Gauze Sponge 10 cm x 10 cm</td><td>Cardinal Health</td><td>361020733</td><td></td></tr><tr><td>Oblong Glass Baking Dish</td><td>Pyrex</td><td>1105397</td><td>Glassware (2.8 L)</td></tr><tr><td>Funnel Buchner</td><td>Coorstek Inc</td><td>10-356E</td><td>Glassware (114 mm diameter)</td></tr><tr><td>Watch Glass&amp;nbsp;</td><td>pyrex</td><td>9985100EMD</td><td>Glassware</td></tr><tr><td>Formalin solution, neutral buffered, 10%</td><td>Sigma-Aldrich</td><td>HT501128-4L</td><td>histological tissue fixative solution</td></tr><tr><td>Trypsinizing Flasks</td><td>Wheaton</td><td>355395</td><td>Glassware (1 unit)</td></tr><tr><td>Disposable Culture Tubes</td><td>Kimble</td><td>73750-13100</td><td>Glassware</td></tr><tr><td>Borosilicate Glass Pasteur Pipet (22.8 cm)</td><td>Fisherbrand</td><td>K63B1367820C</td><td>Glassware</td></tr><tr><td>250 ml Glass Beakers</td><td>Fisherbrand</td><td>KFS14005250</td><td>Glassware</td></tr><tr><td>Glass Media Bottles With Cap</td><td>Fisherbrand</td><td>KFS14395250</td><td>Glassware (8 units)</td></tr><tr><td>50 ml Corex Tube&amp;nbsp;</td><td>Corning</td><td>8422-A</td><td>(1 unit)</td></tr><tr><td>15 ml Polystyrene Centrifuge Tube</td><td>Corning</td><td>430791</td><td></td></tr><tr><td>50 ml Polystyrene Centrifuge Tube</td><td>Corning</td><td>430829</td><td></td></tr><tr><td>10 ml Serological Pipet</td><td>Corning</td><td>11415038</td><td></td></tr><tr><td>Cell Strainer 100 &amp;mu;m Nylon</td><td>Corning</td><td>431752</td><td></td></tr><tr><td>Absorbant Liner</td><td>Scienceware</td><td>1199918</td><td></td></tr><tr><td>500&amp;nbsp;ml Bottles Top Filter</td><td>Corning</td><td></td><td>Pore: 0.22&amp;nbsp;&amp;micro;m / medium and HBSS preparation</td></tr><tr><td>2 ml Criogenic Vials</td><td>Corning</td><td>430488</td><td></td></tr><tr><td>Freezing Container, Nalgene Mr. Frosty</td><td>Sigma-Aldrich</td><td>C1562-1EA</td><td></td></tr><tr><td>Peristaltic Pump</td><td>Pharmacia Fine Chemicals</td><td>P3 model</td><td></td></tr><tr><td>Shaking Water Bath</td><td>Fisher</td><td>Model 127</td><td></td></tr><tr><td>Vacuum Pump</td><td>ABM</td><td>4EKFS6CX-4</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisherbrand</td><td>EC231-598-3</td><td>Saline solution 0.9%</td></tr><tr><td>Hank&amp;rsquo;s Buffered Salt Solution (Hbss)</td><td>Sigma-Aldrich</td><td>H2387</td><td>Quantity: 9.25 (one vial) for 1 L of digestion solution</td></tr><tr><td>Hydroxypiperazineethansulphonic Acid (Hepes)</td><td>&lt;a name=&quot;RANGE!B29&quot;&gt;Life Technologies&lt;/a&gt;</td><td>15630-080</td><td>25 ml (1 M) for 1 L of digestion solution</td></tr><tr><td>Trypsin Type I</td><td>Sigma-Aldrich</td><td>T8003</td><td>9,888 U</td></tr><tr><td>Deoxyribonuclease Type Iv</td><td>Roche</td><td>10-104-159-001</td><td>402,000 U</td></tr><tr><td>Calcium Chloride</td><td>Sigma-Aldrich</td><td>C4901</td><td>100 mM</td></tr><tr><td>Magnesium Sulfate</td><td>Baker</td><td>2500-01</td><td>800 mM</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle Medium High Glucose (Dmem)</td><td>Life Technologies</td><td>10564-045</td><td></td></tr><tr><td>Penicillin/Streptomycin Sulphate</td><td>Hyclone</td><td>SV30010</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Corning</td><td>35-010-CV</td><td></td></tr><tr><td>Percoll</td><td>Sigma-Aldrich</td><td>P1644</td><td>Density centrifugation media gradient. Volume: 36 ml</td></tr><tr><td>Isopropanol</td><td>Acros</td><td>42383-0010</td><td>50 ml</td></tr><tr><td>Dimethyl Sulfoxide</td><td>Sigma-Aldrich</td><td>472301</td><td></td></tr><tr><td>Automacs Magnetic Separator&amp;nbsp;</td><td>Miltenyi Biotec</td><td>Model 003</td><td></td></tr><tr><td>Automacs&amp;nbsp;Columns&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-021-101</td><td></td></tr><tr><td>Automacs Running Buffer&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-221</td><td>http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1</td></tr><tr><td>Automacs Rinsing Solution&amp;nbsp;</td><td>Miltenyi Biotec</td><td>130-091-222</td><td>http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx</td></tr><tr><td>Anti-Human Hla Abc Purified Clone W6/32</td><td>Affymetrix eBioscience</td><td>14-9983-82</td><td>anti-mouse antibody</td></tr><tr><td>Anti Mouse Igg Microbeads</td><td>Miltenyi Biotec</td><td>130048401</td><td></td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 6 Well With Lid</td><td>Corning</td><td>3335</td><td>Cell Bind surface</td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 24 Well With Lid</td><td>Corning</td><td>3337</td><td>Cell Bind surface</td></tr><tr><td>Multiple Well Plate -&amp;nbsp; 96 Well With Lid</td><td>Corning</td><td>3300</td><td>Cell Bind surface</td></tr><tr><td>Modular Incubator Chamber&amp;nbsp;</td><td>Billups-Rothenberg</td><td>MIC-101</td><td>A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions</td></tr><tr><td>Single Flow Meter</td><td>Billups-Rothenberg</td><td>SFM3001</td><td></td></tr><tr><td>50 mm In-Line Filter</td><td>Whatman</td><td>6721-5010</td><td>PTFE, pore: 1.0 &amp;micro;m</td></tr><tr><td>Gas Regulator</td><td>Pro Star</td><td>PRS301233</td><td>A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions</td></tr><tr><td>Gas Hose Class Vi Clear 5/16&amp;nbsp;</td><td>Parker</td><td>100-05070102</td><td>3 pieces with ~ 0.5 m</td></tr><tr><td>17 mm Adjustable Gas Hose Clamp</td><td>Tiewraps</td><td>THCSS-16</td><td></td></tr><tr><td>Normoxia Gas Cylinder&amp;nbsp;</td><td>Praxair</td><td>NI CDOXR1U-K</td><td>Size K (3&lt;sup&gt;rd&lt;/sup&gt; trimester&#039;s composition: 5% CO&lt;sub&gt;2&lt;/sub&gt;, 8% O&lt;sub&gt;2&lt;/sub&gt;, Bal. N&lt;sub&gt;2&lt;/sub&gt;)</td></tr><tr><td>Normoxia Gas Cylinder&amp;nbsp;</td><td>Praxair</td><td>NI CDOXR1U-K</td><td>Size K (3&lt;sup&gt;rd&lt;/sup&gt; trimester&#039;s composition: 5% CO&lt;sub&gt;2&lt;/sub&gt;, 0.5% O&lt;sub&gt;2&lt;/sub&gt;, Bal. N&lt;sub&gt;2&lt;/sub&gt;)</td></tr><tr><td>Oxygen Microelectrode Mi-730</td><td>Microelectrodes INC</td><td>84477</td><td></td></tr><tr><td>Oxygen Adapter</td><td>Microelectrodes INC</td><td>3572</td><td></td></tr><tr><td>ROS Detection Reagent: CM-H2DCFDA&amp;nbsp;</td><td>Invitrogen</td><td>C-400</td><td></td></tr><tr><td>&amp;beta;-hCG ELISA kit&amp;nbsp;</td><td>DRG internatinal</td><td>EIA-4115</td><td></td></tr><tr><td>Anti-Vimentin purified antibody</td><td>eBioscience</td><td>14-9897</td><td>Host: mouse</td></tr><tr><td>Anti-Cytokeratin 7 (FITC) antibody&amp;nbsp;</td><td>Abcam</td><td>ab119697</td><td>Host: mouse</td></tr><tr><td>Alexa Fluor 488 Goat Anti-mousse IgG H&amp;amp;L antibody</td><td>Life Technologies</td><td>A-11029</td><td></td></tr></tbody></table>

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.

Isolation and immunopurification of villous cytotrophoblast cells from a normal term placenta obtained by vaginal delivery yielded 1 x 108 viable cells. The placenta weighed 350 g, was 19 cm in diameter, 4 cm tall with discoid shape and transparent membranes. No cotyledon malformation was detected. The umbilical cord had paracentral localization and a length of 56 cm. The purity was evaluated by flow cytometry using vimentin and cytokeratin-7 markers. More than 98% of the cells were negative for vimentin and positive for cytokeratin-7, confirming the purity of villous trophoblasts cells obtained from the immunopurification. Villous cytotrophoblast cells were added to 96-well culture plates under normoxic conditions in the presence or absence of 1 mM melatonin. The biochemical differentiation of villous cytotrophoblasts was monitored by determining levels of &#946;-human chorionic gonadotropin (&#946;-hCG) secretion as described previously1,7,20,21. The morphological differentiation and apoptosis were assessed by immunofluorescence using anti-desmoplakin and anti-caspase-cleaved cytokeratin 18 intermediate filaments7,22. Cell culture media from day 1 (mainly villous cytotrophoblasts) to day 4 (mainly syncytiotrophoblasts) were collected, centrifuged and &#946;-hCG levels were measured in the supernatants. Production of &#946;-hCG, which is exclusive to the syncytiotrophoblast, increased with culture time (Figure 4). Not only hypoxia/reoxygenation, but hyperoxia (&#62; 20% O2) also activated apoptosis23. Thus, adoption of an 8% O2 concentration was representative of the quantity of oxygen to which a villous trophoblast cell would be exposed during the third trimester of pregnancy10. The peak of &#946;-hCG levels observed at 72 hr confirmed the capacity of villous cytotrophoblasts to differentiate under these conditions. Melatonin did not alter &#946;-hCG secretion under these study conditions. The decrease of &#946;-hCG levels at 96 hr was likely caused by apoptosis of trophoblast cells, which increases after prolonged periods in primary culture5,7,22,24,25 (Figure 4). DMSO (0.1% vol/vol) was selected because it did not affect &#946;-hCG levels26,27. The protective role of melatonin was strongly related to its antioxidant properties. Hypoxia/reoxygenation after 72h of culture induced oxidative stress in villous trophoblast cells. The protective effect of melatonin was assessed with Reactive Oxygen Species (ROS) Detection Reagent (Figure 5A). After 96 hr of culture, trophoblast cells were incubated for 45 min with 10 &#181;M of 5-(and-6)-carboxy-2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) to detect the total amount of ROS produced8. Villous trophoblast cells that underwent hypoxia/reoxygenation had significantly increased ROS levels (54%) compared to those under normoxia. This increase was reversed by treatment with 1 mM melatonin. Moreover, under normoxia melatonin did not modulate ROS levels (homeostasis), which was similar to non-treated villous trophoblast cells (Figure 5A). Figure 4 and 5A show that under normoxia melatonin did not alter levels of oxidative stress or &#946;-hCG secretion in the trophoblast cells, which corroborates previous studies showing no modulation of cell homeostasis under normal conditions28,29.
<img alt="Figure 1" src="/files/ftp_upload/54228/54228fig1.jpg" /> 
Figure 1: Digestion Tube. After centrifugation, 4 layers are formed. The upper layer is composed of digestion solution; just below, the fetal bovine serum (FBS). Both layers should be discarded with a vacuum pump. The lower layers are composed as follow: a white layer containing fibroblasts, leukocytes, macrophages, and trophoblasts; and a bottom layer composed of red blood cells. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54228/54228fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54228/54228fig2.jpg" /> 
Figure 2: Components of the Hypoxia Chamber and Measurement/Calculation of Dissolved Oxygen Concentration. (A) Hypoxia chamber and gas cylinder assembly: (1) Gas cylinder; (2) Gas regulator; (3) Gas hose clamp; (4) Cylinder gas hose; (5) Inlet filter; (6) Inlet hose; (7) Flow meter; (8) Outlet hose; (9) Modular incubator chamber. (B) Calculation of actual oxygen concentration in cell culture medium using a standard curve produced with known oxygen concentrations. (C and D) The relative values obtained in the solutions &#34;0% O2&#34; and &#34;21% O2&#34;, are plotted graphically as a linear function to determine the oxygen concentration in the cell culture medium &#34;?% O2&#34;. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54228/54228fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54228/54228fig3.jpg" /> 
Figure 3: Generic Experimental Design of Cell Culture in the Modular Incubation Chamber. Normoxia (8% O2; 5% CO2; 87% N2) and hypoxia/reoxygenation (H/R) (0.5% O2; 5% CO2; 94.5% N2) are conditions used to study pathological conditions in villous cytotrophoblast (vCTB) and syncytiotrophoblast (STB) cells. Every 24 hr, medium with or without melatonin (1 mM) is changed and the gas mixture is renewed. Under H/R, STB cells undergo hypoxia (0.5% O2) for 4 hr and then return to normoxia (8% O2). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54228/54228fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/54228/54228fig4.jpg" /> 
Figure 4: Effect of Melatonin on beta-human Chorionic Gonadotropin (&#946;-hCG) Secretion during Villous Trophoblast Differentiation. Villous cytotrophoblast cells were isolated and purified from human healthy term placentas. Cells were treated for 96 hr with 1 mM melatonin or dimethyl sulfoxide (DMSO 0.1%: vehicle control) under normoxic conditions (8% O2; 5% CO2; 87% N2). &#946;-hCG levels in culture medium were measured by enzyme-linked immunosorbent assay (ELISA) after 24, 48, 72 and 96 hr of primary culture. Levels were normalized to the protein content of the whole-cell lysate from each corresponding well. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54228/54228fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/54228/54228fig5.jpg" /> 
Figure 5: Anti-oxidant Effect of Melatonin in Syncytiotrophoblast Exposed to Hypoxia/Reoxygenation. (A) The effect of melatonin (M; 1 mM) on intracellular reactive oxygen species (ROS) levels in syncytiotrophoblast cells under normoxia (N) or hypoxia/reoxygenation (HR), induced after 72 hr of culture, was assessed by 5-(and-6)-carboxy-2&#8242;,7&#8242;-dichlorodihydrofluorescein diacetate (carboxy-H2DCFDA) fluorescence. Results are expressed as the mean &#177; SD of 3 different placentas; *** P &#60; 0.001 (Lanoix, et al.8). (B) The cellular pathways involved in the putative protection of melatonin against hypoxia/reoxygenation-induced apoptosis. Primary villous cytotrophoblast cells were cultured for 72 hr under normoxia (8% O2) to allow differentiation into syncytiotrophoblast. Cells were exposed to 1 mM of melatonin or vehicle control and then subjected to hypoxia (0.5% O2) for 4 hr followed by an 18 hr reoxygenation period (8% O2). Hypoxia/reoxygenation-induced oxidative stress activates redox sensitive transcription factors such as nuclear factor kappa B (NF-&#954;B) and hypoxia inducible factor 1 (HIF-1). NF-&#954;B induces p53, which triggers the Bax/Bcl-2 pathway of mitochondrial apoptosis involving the cleavage and activation of caspases 9 and 3. Caspase 3 activates Rho-associated, coiled coil-containing protein kinase 1 (ROCK-1), the cleavage of poly(ADP-ribose) polymerase (PARP) and the impairment of DNA repair. Melatonin prevents the induction of mitochondrial apoptosis by acting as a powerful antioxidant to reduce the oxidative stress caused by hypoxia/reoxygenation. This figure has been modified from Lanoix et al., 20138. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54228/54228fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td></td>
			<td>Digestion 1</td>
			<td>Digestion 2</td>
			<td>Digestion 3</td>
			<td>Digestion 4</td>
		</tr>
		<tr>
			<td>Modified HBSS (ml)</td>
			<td>150</td>
			<td>100</td>
			<td>75</td>
			<td>75</td>
		</tr>
		<tr>
			<td>DNAse (&#181;l)</td>
			<td rowspan="2">300</td>
			<td rowspan="2">200</td>
			<td rowspan="2">150</td>
			<td rowspan="2">150</td>
		</tr>
		<tr>
			<td>(0.1&#160;mg/&#181;l)</td>
		</tr>
		<tr>
			<td>MgSO4 (&#181;l)</td>
			<td rowspan="2">150</td>
			<td rowspan="2">100</td>
			<td rowspan="2">75</td>
			<td rowspan="2">75</td>
		</tr>
		<tr>
			<td>(800 mM)</td>
		</tr>
		<tr>
			<td>CaCl2 (&#181;l)</td>
			<td rowspan="2">150</td>
			<td rowspan="2">100</td>
			<td rowspan="2">75</td>
			<td rowspan="2">75</td>
		</tr>
		<tr>
			<td>(100 mM)</td>
		</tr>
		<tr>
			<td>Trypsin (U)</td>
			<td>1,824,000</td>
			<td>1,200,000</td>
			<td>960,000</td>
			<td>960,000</td>
		</tr>
		<tr>
			<td>P/S (ml)</td>
			<td>1</td>
			<td>0</td>
			<td>0</td>
			<td>0</td>
		</tr>
	</tbody>
</table>
Table 1: Quantities of Ingredients for the Digestion Solution. Penicillin and streptomycin (P/S); magnesium sulfate (MgSO4); calcium chloride (CaCl2); deoxyribonuclease IV (DNase IV).
<img alt="Table 2" src="/files/ftp_upload/54228/54228table2.jpg" /> 
Table 2: Volumes of Density Centrifugation Media Solution and Modified HBSS Required for Preparation of the Gradient Solution.

Watch this Scientific Journal Video about Human Primary Trophoblast Cell Culture Model to Study the Protective Effects of Melatonin Against Hypoxia/reoxygenation-induced Disruption at JoVE.com

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

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 ...

human-primary-trophoblast-cell-culture-model-to-study-protective

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

15.8K Views.  INRS-Institut Armand-Frappier. 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.

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

Experimental Models for Study of Retinal Pigment Epithelial Physiology and Pathophysiology

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium (hfRPE) cultures with morphological, physiological and genetic characteristics of native human RPE. These hfRPE cell cultures exhibit heavy pigmentation, and electron microscopy show extensive apical membrane microvilli. The junctional complexes were identified with immunofluorescence labeling of various tight junction proteins. Epithelial polarity and function of these easily reproducible primary cultures closely resemble previously studied mammalian models of native RPE, including human. These results were extended by the development of therapeutic interventions in several animal models of human eye disease. We have focused on strategies for the removal of abnormal fluid accumulation in the retina or subretinal space. The extracellular subretinal space separates the photoreceptor outer segments and the apical membrane of the RPE and is critical for maintenance of retinal attachments and a whole host of RPE/retina interactions.

We provide a reproducible method for culturing confluent monolayers of human fetal retinal pigment epithelial cells (hfRPE) cells that exhibit morphology, physiology, polarity, and protein and gene expression patterns of adult native tissue. This work has been extended to an animal model of several eye diseases.

We have developed a cell culture procedure that can produce large quantities of confluent monolayers of primary human fetal retinal pigment epithelium ...

Neuroscience

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

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

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

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

Bioengineering

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

In Vitro Differentiation of Human Pluripotent Stem Cells into Trophoblastic Cells

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

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

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 ...

Translational 3D-Cell Culture Model to Assess Hyperoxia Effects on Human Neonatal Airway Epithelial Cells

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes oxygen (O2) tension higher than the ambient atmosphere - termed hyperoxia (&gt;21% O2). The effect of hyperoxia on the airway depends on various factors, including the developmental stage of the airway, the degree of hyperoxia, and the duration of exposure, with variable exposures potentially leading to unique phenotypes. While there has been extensive research on the effect of hyperoxia on neonatal lung alveolarization and airway hyperreactivity, little is known about the short and long-term underlying effect of hyperoxia on human neonatal airway epithelial cells. A major reason for this is the scarcity of an effective in vitro model to study human neonatal airway epithelial development and function. Here, we describe a method for isolating and expanding human neonatal tracheal airway epithelial cells (nTAECs) utilizing human neonatal tracheal aspirates and culturing these cells in air-liquid interface (ALI) culture. We demonstrate that nTAECs form a mature polarized cell-monolayer in ALI culture and undergo mucociliary differentiation. We also present a method for moderate hyperoxia exposure of the cell monolayer in ALI culture using a specialized incubator. Additionally, we describe an assay to measure cellular oxidative stress following hyperoxia exposure in ALI culture using fluorescent quantification, which confirms that moderate hyperoxia exposure induces cellular oxidative stress but does not cause significant cell membrane damage or apoptosis. This model can potentially be used to simulate clinically relevant hyperoxia exposure encountered by neonatal airways in the Neonatal Intensive Care Unit (NICU) and used to study the short and long-lasting effects of O2 on neonatal airway epithelial programming. Studies using this model could be utilized to explore ways to mitigate early-life oxidative injury to developing airways, which is implicated in the development of long-term airway diseases in former premature infants.

We describe a protocol to establish an air-liquid interface (ALI) culture model utilizing neonatal tracheal airway epithelial cells (nTAEC) and perform physiologically relevant hyperoxia exposure to study the effect of atmospheric-induced oxidative stress on cells derived from the developing neonatal airway surface epithelium.

The preterm neonatal airway epithelium is constantly exposed to environmental stressors. One of these stressors in neonates with lung disease includes ...

Isolation of Primary Mouse Trophoblast Cells and Trophoblast Invasion Assay

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes. 
Thus, defects in placental development have important consequences for the fetus and mother, and are a major cause of embryonic lethality. The major cell type of the 
fetal portion of the placenta is the trophoblast. Primary mouse placental trophoblast cells are a useful tool for studying normal and abnormal placental development, 
and unlike cell lines, may be isolated and used to study trophoblast at specific stages of pregnancy. In addition, primary cultures of trophoblast from transgenic
mice may be used to study the role of particular genes in placental cells. The protocol presented here is based on the description by Thordarson et al.1, in which a 
percoll gradient is used to obtain a relatively pure trophoblast cell population from isolated mouse placentas. It is similar to the more widely used methods for 
human trophoblast cell isolation2-3. Purity may be assessed by immunocytochemical staining of the isolated cells for cytokeratin 74. Here, the isolated cells are 
then analyzed using a matrigel invasion assay to assess trophoblast invasiveness in vitro5-6. The invaded cells are analyzed by immunocytochemistry and stained 
for counting.

In this protocol, we describe the dissection of placentae from the mouse on pregnancy d10.5, followed by isolation of trophoblast cells using a Percoll gradient. We then demonstrate use of the isolated cells in a matrigel invasion assay.

The placenta is responsible for the transport of nutrients, gasses and growth factors to the fetus, as well as the elimination of wastes.  
Thus, defects ...

Biology

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

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

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

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

Medicine

In vitro Assessment of Myocardial Protection following Hypothermia-Preconditioning in a Human Cardiac Myocytes Model

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of cardiomyocytes to ischemic injury is higher than that of other cell populations. At present, hypothermia affords considerable protection against an expected ischemic insult. However, investigations into complex hypothermia-induced molecular changes remain limited. Therefore, it is essential to identify a culture condition similar to in vivo conditions that can induce damage similar to that observed in the clinical condition in a reproducible manner. To mimic ischemia-like conditions in vitro, the cells in these models were treated by oxygen/glucose deprivation (OGD). In addition, we applied a standard time-temperature protocol used during cardiac surgery. Furthermore, we propose an approach to use a simple but comprehensive method for the quantitative analysis of myocardial injury. Apoptosis and expression levels of apoptosis-associated proteins were assessed by flow cytometry and using an ELISA kit. In this model, we tested a hypothesis regarding the effects of different temperature conditions on cardiomyocyte apoptosis in vitro. The reliability of this model depends on strict temperature control, controllable experimental procedures, and stable experimental results. Additionally, this model can be used to study the molecular mechanism of hypothermic cardioprotection, which may have important implications for the development of complementary therapies for use with hypothermia.

The distinct effects of different degrees of hypothermia on myocardial protection have not been thoroughly evaluated. The goal of the present study was to quantify the levels of cell death following different hypothermia treatments in a human cardiomyocyte-based model, laying the foundation for future in-depth molecular research.

Ischemia/reperfusion-derived myocardial dysfunction is a common clinical scenario in patients after cardiac surgery. In particular, the sensitivity of ...

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 million patients worldwide. It leads to a decrease in retinal pigment epithelium (RPE)-synthesized neuroprotective factors, e.g., pigment epithelium-derived factor (PEDF) and granulocyte-macrophage colony-stimulating factor (GM-CSF), followed by the loss of RPE cells, and eventually photoreceptor and retinal ganglion cell (RGC) death. We hypothesize that the reconstitution of the neuroprotective and neurogenic retinal environment by the subretinal transplantation of transfected RPE cells overexpressing PEDF and GM-CSF has the potential to prevent retinal degeneration by mitigating the effects of oxidative stress, inhibiting inflammation, and supporting cell survival. Using the Sleeping Beauty transposon system (SB100X) human RPE cells have been transfected with the PEDF and GM-CSF genes and shown stable gene integration, long-term gene expression, and protein secretion using qPCR, western blot, ELISA, and immunofluorescence. To confirm the functionality and the potency of the PEDF and GM-CSF secreted by the transfected RPE cells, we have developed an in vitro assay to quantify the reduction of H2O2-induced oxidative stress on RPE cells in culture. Cell protection was evaluated by analyzing cell morphology, density, intracellular level of glutathione, UCP2 gene expression, and cell viability. Both, transfected RPE cells overexpressing PEDF and/or GM-CSF and cells non-transfected but pretreated with PEDF and/or GM-CSF (commercially available or purified from transfected cells) showed significant antioxidant cell protection compared to non-treated controls. The present H2O2-model is a simple and effective approach to evaluate the antioxidant effect of factors that may be effective to treat AMD or similar neurodegenerative diseases.

Induction and Analysis of Oxidative Stress in Sleeping Beauty Transposon-Transfected Human Retinal Pigment Epithelial Cells

We present a protocol for the development and use ofan oxidative stress-model by treating retinal pigment epithelial cells with H2O2, analyzing cell morphology, viability, density, glutathione, and UCP-2 level. It is a useful model to investigate the antioxidant effect of proteins secreted by transposon-transfected cells to treat neuroretinal degeneration.

Oxidative stress plays a critical role in several degenerative diseases, including age-related macular degeneration (AMD), a pathology that affects ~30 ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...

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

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