Method Article

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

DOI:

10.3791/54228

⸱

July 30th, 2016

 ,  ,  ,  ,  ,  ,  ,  ,  , 
1INRS-Institut Armand-Frappier

* These authors contributed equally

In This Article

Summary

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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.

Abstract

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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.

Introduction

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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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Protocol

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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

  1. Solutions and media
    1. Prepare transport media by supplementing Dulbecco's Modified Eagle'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 °C.
    2. 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 °C. Warm media to 37 °C before use.
    3. Prepare 4 L of saline solution (0.9% weight/vol sodium chloride).
    4. Prepare modified Hank's Balanced Salt Solution (HBSS) by adding 25 mM HEPES to 1x HBSS (pH 7.4).
    5. 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.
    6. Prepare density centrifugation media gradient
      1. Prepare density centrifugation media solution by supplementing density centrifugation media with 10% vol/vol HBSS 10x.
      2. Prepare 14 assay tubes with density centrifugation media solution and modified HBSS, as described in Table 2.
      3. 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.
        1. In absence of a peristaltic pump, apply the layers very gently with Pasteur pipettes.
    7. 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 °C.
  2. Villous cytotrophoblast isolation
    Note: Use sterile surgical equipment, glassware, pipettes, flasks, etc.
    1. On the day of villous cytotrophoblast isolation, in a 37 °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 (1.2.22) which can be placed on ice once thawed.
    2. 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).
    3. Discard transport medium and placental blood in liquid dustbin. Weigh the placenta and immerse in cold saline solution.
    4. 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.
    5. 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.
    6. 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.
    7. 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.
      1. Place minced placenta in a Büchner 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.
    8. 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.
    9. 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.
    10. At the end of the first digestion, remove the trypsinizing flask from the water bath and tilt it (45°) 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.
    11. Transfer 100 ml of digestion solution 2 (Table 1) to the trypsinizing flask; mix well and repeat step 1.2.9.
    12. At the end of the second digestion, remove the trypsinizing flask from the water bath and tilt it 45° 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 µm mesh). Transfer the filtered supernatant to a beaker containing 2 ml of FBS every time the centrifuge tube is full.
    13. 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.
    14. 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.
    15. 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.
    16. 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).
      1. 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).
    17. 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.
    18. 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.
    19. 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.
    20. 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 (> 50%).
    21. 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.
    22. 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 °C and transfer to a liquid nitrogen tank.
  3. Trophoblast purification
    1. Install the rinsing and running buffers and a new filter column on the magnetic purification instrument according to manufacturer's instructions. Perform the "clean program" to clean negative 1, positive 1 and positive 2 ports, and then introduce the 50 ml tubes under each port according to manufacturer's instructions.
    2. Thaw the cells that were frozen in step 1.2.22 quickly in a 37 °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 °C.
    3. 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 °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 °C for 30 min, mixing gently every 5 min.
    4. Add 6 ml of cold running buffer. Centrifuge for 5 min at 450 x g and 4 °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 °C for 15 min, mixing gently every 5 min.
    5. Add 6 ml of cold running buffer. Centrifuge for 5 min at 450 x g and 4 °C. Discard the supernatant and resuspend in 5 ml cold running buffer.
    6. 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.
    7. Centrifuge for 5 min at 450 x g and 4 °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.
    8. 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 °C and 5% CO2.
    9. 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.
    10. 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).

2. In Vitro Induction of Normoxia and Hypoxia/Reoxygenation

  1. Incubator chamber operation (see Figure 2A for set-up).
    1. 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.
    2. 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.
    3. 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.
    4. After flushing the chamber, close the gas regulator then the inlet and outlet ports of the chamber.
    5. 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 °C.
    6. 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.
    7. Repeat steps 2.1.1 to 2.1.6 for the other gas compositions (e.g., 2% O2 for first trimester trophoblast culture9).
  2. Confirm the oxygen percentage (Figure 2B-C)
    1. 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.
    2. 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.
  3. Induction of normoxia and hypoxia/reoxygenation in trophoblasts.
    1. After adding primary trophoblast cells to the appropriate cell culture flasks, plates or Petri dishes, perform treatments as necessary.
    2. In parallel, inside the chamber, expose cells to the desired gas mixture to reproduce a specific condition every 24 hr (Figure 3).

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Results

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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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Discussion

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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...

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Disclosures

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The authors have nothing to disclose.

Acknowledgements

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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 Ministère de l'éducation, de l'Enseignement supérieurs et de la recherche (MEESR)-Fonds de recherche du Québec (FRQ)-Nature et technologies (NT) and the Fondation Universitaire Armand-Frappier INRS, to HC from the Réseau Québécois en Reproduction-NSERC-CREATE, to AAHT from the Canadian Institutes of Health Research (CIHR) and FRQ-Santé, and to JBP from NSERC; by a fellowship to EMAS from the Conselho Nacional de Desenvolvimento Cientìfico e Tecnològico (CNPq) and the Programme de bourses d'excellence pour étudiants étrangers MEESR-FRQNT.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Curved Metzenbaum ScissorsShandon9212surgical equipment (cell isolation) (2 units)
Splinter Forceps Fine 41/2 inFisherbrand13-812-42surgical equipment (cell isolation) (2 units)
Scissors 4.5 Str DissectionFisherbrand08-940surgical equipment (cell isolation) (2 units)
Gauze Sponge 10 cm x 10 cmCardinal Health361020733
Oblong Glass Baking DishPyrex1105397Glassware (2.8 L)
Funnel BuchnerCoorstek Inc10-356EGlassware (114 mm diameter)
Watch Glass pyrex9985100EMDGlassware
Formalin solution, neutral buffered, 10%Sigma-AldrichHT501128-4Lhistological tissue fixative solution
Trypsinizing FlasksWheaton355395Glassware (1 unit)
Disposable Culture TubesKimble73750-13100Glassware
Borosilicate Glass Pasteur Pipet (22.8 cm)FisherbrandK63B1367820CGlassware
250 ml Glass BeakersFisherbrandKFS14005250Glassware
Glass Media Bottles With CapFisherbrandKFS14395250Glassware (8 units)
50 ml Corex Tube Corning8422-A(1 unit)
15 ml Polystyrene Centrifuge TubeCorning430791
50 ml Polystyrene Centrifuge TubeCorning430829
10 ml Serological PipetCorning11415038
Cell Strainer 100 μm NylonCorning431752
Absorbant LinerScienceware1199918
500 ml Bottles Top FilterCorningPore: 0.22 µm / medium and HBSS preparation
2 ml Criogenic VialsCorning430488
Freezing Container, Nalgene Mr. FrostySigma-AldrichC1562-1EA
Peristaltic PumpPharmacia Fine ChemicalsP3 model
Shaking Water BathFisherModel 127
Vacuum PumpABM4EKFS6CX-4
Sodium ChlorideFisherbrandEC231-598-3Saline solution 0.9%
Hank’s Buffered Salt Solution (Hbss)Sigma-AldrichH2387Quantity: 9.25 (one vial) for 1 L of digestion solution
Hydroxypiperazineethansulphonic Acid (Hepes)Life Technologies15630-08025 ml (1 M) for 1 L of digestion solution
Trypsin Type ISigma-AldrichT80039,888 U
Deoxyribonuclease Type IvRoche10-104-159-001402,000 U
Calcium ChlorideSigma-AldrichC4901100 mM
Magnesium SulfateBaker2500-01800 mM
Dulbecco’s Modified Eagle Medium High Glucose (Dmem)Life Technologies10564-045
Penicillin/Streptomycin SulphateHycloneSV30010
Fetal Bovine SerumCorning35-010-CV
PercollSigma-AldrichP1644Density centrifugation media gradient. Volume: 36 ml
IsopropanolAcros42383-001050 ml
Dimethyl SulfoxideSigma-Aldrich472301
Automacs Magnetic Separator Miltenyi BiotecModel 003
Automacs Columns Miltenyi Biotec130-021-101
Automacs Running Buffer Miltenyi Biotec130-091-221http://www.miltenyibiotec.com/~/media/Images/Products/Import/0001100/IM0001131.ashx?force=1
Automacs Rinsing Solution Miltenyi Biotec130-091-222http://www.miltenyibiotec.com/en/products-and-services/macs-cell-separation/cell-separation-buffers/automacs-rinsing-solution.aspx
Anti-Human Hla Abc Purified Clone W6/32Affymetrix eBioscience14-9983-82anti-mouse antibody
Anti Mouse Igg MicrobeadsMiltenyi Biotec130048401
Multiple Well Plate -  6 Well With LidCorning3335Cell Bind surface
Multiple Well Plate -  24 Well With LidCorning3337Cell Bind surface
Multiple Well Plate -  96 Well With LidCorning3300Cell Bind surface
Modular Incubator Chamber Billups-RothenbergMIC-101A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Single Flow MeterBillups-RothenbergSFM3001
50 mm In-Line FilterWhatman6721-5010PTFE, pore: 1.0 µm
Gas RegulatorPro StarPRS301233A set of two is necessary for simultaneous to generate normoxia and hypoxia/reoxygenation conditions
Gas Hose Class Vi Clear 5/16 Parker100-050701023 pieces with ~ 0.5 m
17 mm Adjustable Gas Hose ClampTiewrapsTHCSS-16
Normoxia Gas Cylinder PraxairNI CDOXR1U-KSize K (3rd trimester's composition: 5% CO2, 8% O2, Bal. N2)
Normoxia Gas Cylinder PraxairNI CDOXR1U-KSize K (3rd trimester's composition: 5% CO2, 0.5% O2, Bal. N2)
Oxygen Microelectrode Mi-730Microelectrodes INC84477
Oxygen AdapterMicroelectrodes INC3572
ROS Detection Reagent: CM-H2DCFDA InvitrogenC-400
β-hCG ELISA kit DRG internatinalEIA-4115
Anti-Vimentin purified antibodyeBioscience14-9897Host: mouse
Anti-Cytokeratin 7 (FITC) antibody Abcamab119697Host: mouse
Alexa Fluor 488 Goat Anti-mousse IgG H&L antibodyLife TechnologiesA-11029

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Villous Cytotrophoblast IsolationPrimary Trophoblast CultureHypoxia Reoxygenation ModelMelatonin Protective EffectsImmunomagnetic PurificationDensity CentrifugationFlow Cytometry AnalysisSyncytiotrophoblast DifferentiationOxidative Stress AssessmentBeta hCG Secretion

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