Name: human primary trophoblast cell culture model to study the protective effects of melatonin against hypoxia/reoxygenation-induced disruption
Uploaded: 2016-07-30T15:00:00
Description: 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

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% ...

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.

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			<td>15 Ml Polystyrene Centrifuge Tube</td>
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			<td>430829</td>
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			<td>Cell Strainer 100&#956;m Nylon</td>
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			<td>2 Ml Criogenic Vials</td>
			<td>Corning</td>
			<td>430488</td>
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			<td>Freezing Container, Nalgene Mr. Frosty</td>
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			<td>Peristaltic Pump</td>
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			<td>Hank&#8217;s Buffered Salt Solution (Hbss)</td>
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			<td>Hydroxypiperazineethansulphonic Acid (Hepes)</td>
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			<td>Dimethyl Sulfoxide</td>
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			<td>Automacs Rinsing Solution&#160;</td>
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			<td>Multiple Well Plate -&#160; 6 Well With Lid</td>
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			<td>Multiple Well Plate -&#160; 24 Well With Lid</td>
			<td>Corning</td>
			<td>3337</td>
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			<td>Multiple Well Plate -&#160; 96 Well With Lid</td>
			<td>Corning</td>
			<td>3300</td>
			<td>Cell Bind surface</td>
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			<td>Modular Incubator Chamber&#160;</td>
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			<td>MIC-101</td>
			<td>A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions</td>
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			<td>Single Flow Meter</td>
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			<td>50 Mm In-Line Filter&#160;</td>
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			<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>
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			<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>
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			<td>17 Mm Adjustable Gas Hose Clamp</td>
			<td>Tiewraps</td>
			<td>THCSS-16</td>
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			<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>
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		<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>
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			<td>Oxygen Microelectrode Mi-730</td>
			<td>Microelectrodes INC</td>
			<td>84477</td>
			<td></td>
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			<td>Oxygen Adapter</td>
			<td>Microelectrodes INC</td>
			<td>3572</td>
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			<td>ROS Detection Reagent: CM-H2DCFDA&#160;</td>
			<td>Invitrogen</td>
			<td>C-400</td>
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			<td>&#946;-hCG ELISA kit&#160;</td>
			<td>DRG internatinal</td>
			<td>EIA-4115</td>
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			<td>Anti-Vimentin ourified antibody</td>
			<td>eBioscience</td>
			<td>14-9897</td>
			<td>Host: mouse</td>
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		<tr>
			<td>Anti-Cytokeratin 7 (FITC) antibody&#160;</td>
			<td>Abcam</td>
			<td>ab119697</td>
			<td>Host: mouse</td>
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		<tr>
			<td>Alexa Fluor 488 Goat Anti-mousse IgG H&#38;L antibody</td>
			<td>Life Technologies</td>
			<td>A-11029</td>
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	</tbody>
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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...

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 ...

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