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 Klima...

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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 41/2in</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 10cm X 10cm</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 (2.8L)</td>
		</tr>
		<tr>
			<td>Funnel Buchner&#160;</td>
			<td>Coorstek Inc</td>
			<td>10-356E</td>
			<td>Glassware (114MM DIAMeter)</td>
		</tr>
		<tr>
			<td>Watch Glass&#160;</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)&#160;</td>
			<td>Fisherbrand</td>
			<td>K63B1367820C</td>
			<td>Glassware</td>
		</tr>
		<tr>
			<td>250 Ml Glass Beakers&#160;</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&#160;</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>10ml Serological Pipet</td>
			<td>Corning</td>
			<td>11415038</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Strainer 100&#956;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&#160;Ml Bottles Top Filter&#160;</td>
			<td>Corning</td>
			<td></td>
			<td>Pore: 0,22&#160;&#181;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&#8217;s Buffered Salt Solution (Hbss)</td>
			<td>Sigma-Aldrich</td>
			<td>H2387</td>
			<td>Quantity: 9.25 (one vial) for 1L of digestion solution</td>
		</tr>
		<tr>
			<td>Hydroxypiperazineethansulphonic Acid (Hepes)</td>
			<td><a name="RANGE!B29">Life Technologies</a></td>
			<td>15630-080</td>
			<td>25mL (1M) for 1L of digestion solution</td>
		</tr>
		<tr>
			<td>Trypsin Type I</td>
			<td>Sigma-Aldrich</td>
			<td>T8003</td>
			<td>9,888U</td>
		</tr>
		<tr>
			<td>Deoxyribonuclease Type Iv</td>
			<td>Roche</td>
			<td>10-104-159-001</td>
			<td>402,000U</td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma-Aldrich</td>
			<td>C4901</td>
			<td>100mM</td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Baker</td>
			<td>2500-01</td>
			<td>800mM</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;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>&#160;Density centrifugation media gradient. Volume: 36mL</td>
		</tr>
		<tr>
			<td>Isopropanol</td>
			<td>Acros</td>
			<td>42383-0010</td>
			<td>50mL</td>
		</tr>
		<tr>
			<td>Dimethyl Sulfoxide</td>
			<td>Sigma-Aldrich</td>
			<td>472301</td>
			<td></td>
		</tr>
		<tr>
			<td>Automacs Magnetic Separator&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>Model 003</td>
			<td></td>
		</tr>
		<tr>
			<td>Automacs&#160;Columns&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td></td>
		</tr>
		<tr>
			<td>Automacs Running Buffer&#160;</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&#160;</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 -&#160; 6 Well With Lid</td>
			<td>Corning</td>
			<td>3335</td>
			<td>Cell Bind surface</td>
		</tr>
		<tr>
			<td>Multiple Well Plate -&#160; 24 Well With Lid</td>
			<td>Corning</td>
			<td>3337</td>
			<td>Cell Bind surface</td>
		</tr>
		<tr>
			<td>Multiple Well Plate -&#160; 96 Well With Lid</td>
			<td>Corning</td>
			<td>3300</td>
			<td>Cell Bind surface</td>
		</tr>
		<tr>
			<td>Modular Incubator Chamber&#160;</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&#160;</td>
			<td>Whatman</td>
			<td>6721-5010</td>
			<td>PTFE, pore: 1.0 &#181;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&#160;</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&#160;</td>
			<td>Praxair</td>
			<td>NI CDOXR1U-K</td>
			<td>Size K (3rd trimester&#8216;s composition: 5% CO2, 8% O2, Bal. N2)</td>
		</tr>
		<tr>
			<td>Normoxia Gas Cylinder&#160;</td>
			<td>Praxair</td>
			<td>NI CDOXR1U-K</td>
			<td>Size K (3rd trimester&#8216;s composition: 5% CO2, 0.5% O2, Bal. N2)</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&#160;</td>
			<td>Invitrogen</td>
			<td>C-400</td>
			<td></td>
		</tr>
		<tr>
			<td>&#946;-hCG ELISA kit&#160;</td>
			<td>DRG internatinal</td>
			<td>EIA-4115</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-Vimentin ourified antibody</td>
			<td>eBioscience</td>
			<td>14-9897</td>
			<td>Host: mouse</td>
		</tr>
		<tr>
			<td>Anti-Cytokeratin 7 (FITC) antibody&#160;</td>
			<td>Abcam</td>
			<td>ab119697</td>
			<td>Host: mouse</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 Goat Anti-mousse IgG H&#38;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...

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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

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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

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated from human muscle biopsy samples using enzymatic digestion and their myogenic properties studied in culture. Quantitatively, the two main adherent cell types obtained from enzymatic digestion are: (i) the satellite cells (termed myogenic cells or muscle precursor cells), identified initially as CD56+ and later as CD56+/desmin+ cells and (ii) muscle-derived fibroblasts, identified as CD56&#8211; and TE-7+. Fibroblasts proliferate very efficiently in culture and in mixed cell populations these cells may overrun myogenic cells to dominate the culture. The isolation and purification of different cell types from human muscle is thus an important methodological consideration when trying to investigate the innate behavior of either cell type in culture. Here we describe a system of sorting based on the gentle enzymatic digestion of cells using collagenase and dispase followed by magnetic activated cell sorting (MACS) which gives both a high purity (&#62;95% myogenic cells) and good yield (~2.8 x 106 &#177; 8.87 x 105 cells/g tissue after 7 days in vitro) for experiments in culture. This approach is based on incubating the mixed muscle-derived cell population with magnetic microbeads beads conjugated to an antibody against CD56 and then passing cells though a magnetic field. CD56+ cells bound to microbeads are retained by the field whereas CD56&#8211; cells pass unimpeded through the column. Cell suspensions from any stage of the sorting process can be plated and cultured. Following a given intervention, cell morphology, and the expression and localization of proteins including nuclear transcription factors can be quantified using immunofluorescent labeling with specific antibodies and an image processing and analysis package.

The main adherent cell types derived from human muscle are myogenic cells and fibroblasts. Here, cell populations are enriched using magnetic-activated cell sorting based on the CD56 antigen. Subsequent immunolabelling with specific antibodies and use of image analysis techniques allows quantification of cytoplasmic and nuclear characteristics in individual cells.

The repair and regeneration of skeletal muscle requires the action of satellite cells, which are the resident muscle stem cells. These can be isolated ...

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head and heart progenitor cell development with pharyngeal arches (also known as branchial arches) ex vivo. Using this method, we have recently described that the second pharyngeal arch contains self-renewing heart progenitors and serves as a microenvironment for expansion of the progenitors during mouse heart development. The progenitor cells remain undifferentiated and expansive inside the arch, but quickly become functional cardiomyocytes as they migrate out of the arch. We also reported that first pharyngeal arch contains muscle progenitors giving rise to myotubes after leaving the arch. Here, we demonstrate the procedure for the dissection and ex vivo culture of first and second pharyngeal arches from developing mouse embryos. The method enables one to study head and heart progenitor/muscle development, including cardiomyocyte and myotube formation in detail ex vivo.

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Here, we present a protocol to culture pharyngeal arches to study the biology of heart and muscle progenitor cells and their microenvironment.

The pharyngeal mesoderm of developing embryos contributes to broad regions of head and heart musculature. We have developed a novel method to study head ...

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 ...

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient utilization by avian embryos during development. Fertilized Japanese quail eggs were incubated at 37 &#176;C for 5 days and then yolk sac membranes (YSM) were collected to establish the EEC culture system. We isolated the embryonic endoderm layer from YSM, and sliced the membrane into 2 - 3 mm pieces and partially digested with collagenase before seeding in 24-well culture plates. The EECs proliferate out of the tissue and are ready for cell culture studies. We found that the EECs had typical characteristics of YSM in vivo, for example, accumulation of lipid droplets, expression of sterol O-acyltransferase and lipoprotein lipase. The partial digestion treatment significantly increased the successful rate of EEC culture. Utilizing the EECs, we demonstrated that the expression of SOAT1 was regulated by the cAMP dependent protein kinase A related pathway. This primary Japanese quail EEC culture system is a useful tool to study embryonic lipid transportation and to clarify the role of genes involved in mediating nutrient utilization in YSM during avian embryonic development.

To study the mechanism of lipid utilization in yolk sac membranes during the late stages of avian embryonic development, we established a primary Japanese quail embryonic endodermal epithelial cell culture system.

We established an endodermal epithelial cell culture model (EEC) for studying the function of certain enzymes and proteins in mediating nutrient ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in the elderly and end-stage renal disease (ESRD) patients, who commonly suffer from hyperphosphatemia and hypercalcemia. At present, there are no medication therapies that can stop its progression. The mechanisms that underlie this pathological process remain unclear. The aortic valve leaflet is composed of a thin layer of valve endothelial cells (VECs) on the outer surfaces of the aortic cusps, with valve interstitial cells (VICs) sandwiched between the VECs. The use of a rat model enables the in vitro study of ectopic calcification based on the in vivo physiopathological serum phosphate (Pi) and calcium (Ca) levels of patients who suffer from hyperphosphatemia and hypercalcemia. The described protocol details the isolation of a pure rat VIC population as shown by the expression of VIC markers: alpha-smooth muscle actin (&#945;-SMA) vimentin and tissue growth factor beta (TGF&#946;) 1 and 2, and the absence of cluster of differentiation (CD) 31, a VEC marker. By expanding these VICs, biochemical, genetic, and imaging studies can be performed to study and unravel the key mediators underpinning CAVD.

This protocol describes the isolation, culture, and calcification of rat-derived valve interstitial cells, a highly physiological in vitro model of calcific aortic valve disease (CAVD). Exploitation of this rat model facilitates CAVD research in exploring the cell and molecular mechanisms that underlie this complex pathological process.

Calcific aortic valve disease (CAVD) is characterized by the progressive thickening of the aortic valve leaflets. It is a condition frequently found in ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

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

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Chapters in this video

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Isolation and Quantitative Immunocytochemical Characterization of Primary Myogenic Cells and Fibroblasts from Human Skeletal Muscle

Ex Vivo Culture of Pharyngeal Arches to Study Heart and Muscle Progenitors and Their Niche

Assessment of Maternal Vascular Remodeling During Pregnancy in the Mouse Uterus

Primary Endodermal Epithelial Cell Culture from the Yolk Sac Membrane of Japanese Quail Embryos

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Using Primary Neurosphere Cultures to Study Primary Cilia

Isolation and Characterization of Primary Rat Valve Interstitial Cells: A New Model to Study Aortic Valve Calcification

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues