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Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Published: June 18th, 2013



1Department of Obstetrics, Perinatal Pharmacology, University Hospital Zurich, 2Laboratory for Materials - Biology Interactions, EMPA Swiss Federal Laboratories for Materials Testing and Research, 3Graduate School for Cellular and Biomedical Sciences, University of Bern

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.

Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.

The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

The placenta is a complex organ which is responsible for the exchange of oxygen, carbon dioxide, nutrients and waste products and at the same time able to keep the two blood circuits of the mother and the growing fetus separated from each other. Additionally, it prevents rejection of the child by the maternal immune system and secretes hormones to maintain pregnancy. The cellular barrier is formed by the cytotrophoblast cells which fuse and form a true syncytium without lateral cell membranes 4,5. The whole placenta is organized in several cotyledons, which contain one fetal villous tree and represent one functional unit of the placenta.

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1. Preparing the Perfusion System

  1. Set up the perfusion system consisting of a water bath, a perfusion chamber, two columns for oxygenation, two peristaltic pumps, two bubble traps, two flow heaters and one pressure sensor (Figure 1). Connect these components with tubing sections composed of silicone and polyvinyl chloride materials according to the scheme in Figure 2. Finally there are two circuits representing the fetal and maternal circuit, respectively.
  2. Turn on t.......

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Figure 4A shows the perfusion profiles of small polystyrene particles (80 nm) which were transported across the placenta compared to bigger polystyrene particles (500 nm) which were not transferred to the fetal compartment. Each data point represents the mean particle concentration to the given time point of at least 3 independent experiments. For polystyrene nanoparticles the placental transfer is size-dependent 19. After 3 hr of placenta perfusion already 20-30% of the initially added 80 nm .......

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Beneath the dual recirculating perfusion showed here, there are several other experimental configurations possible depending on the question which has to be answered. Particularly open placental perfusions are commonly used to assess the drug clearance at steady-state concentration 3. The recirculating perfusion set-up can be also applied to confirm active transport of endogenous or exogenous substances. For this approach the same concentration of the xenobiotic has to be added to the maternal and the fetal ci.......

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This work is financially supported by the Swiss National Foundation, (NRP 64 program, grant no 4064-131232).


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Name Company Catalog Number Comments
Name of the Reagent Company Catalogue Number Comments (optional)
NCTC-135 medium ICN Biomedicals, Inc. 10-911-22C could be replaced by Medium 199 from Sigma (M3769)
Sodium chloride (NaCl) Sigma-Aldrich, Fluka 71381
Potassium chloride (KCl) Hospital pharmacy also possible: Sigma (P9541)
Monosodium phosphate (NaH2PO4 · H2O) Merck 106346
Magnesium sulfate (MgSO4 · H2O) Sigma-Aldrich, Fluka 63139
Calcium chloride (CaCl, anhydrous) Merck 102388
D(+) Glucose (anhydrous) Sigma-Aldrich, Fluka 49138
Sodium bicarbonate (NaHCO3) Merck 106329
Dextran from Leuconostoc spp. Sigma-Aldrich 31389
Bovine serum albumin (BSA) Applichem A1391
Amoxicilline (Clamoxyl) GlaxoSmithKline AG 2021101A
Sodium heparin B. Braun Medical AG 3511014
Sodium hydoxide (NaOH) pellets Merck 106498 CAUTION: corrosive
Ortho-phosphoric acid 85% (H3PO4) Merck 100573 CAUTION: corrosive
Maternal gas mixture: 95% synthetic air, 5% CO2 PanGas AG
Fetal gas mixture: 95% N2, 5% CO2 PanGas AG
Antipyrine (N-methyl-14C) American Radiolabeled Chemicals, Inc. ARC 0108-50 μCi CAUTION: radioactive material (specific activity: 55mCi/mmol)
Scintillation cocktail (IrgaSafe Plus) Zinsser Analytic GmbH 1003100
Polystyrene particles 80 nm Polyscience, Inc. 17150
Polystyrene particles 500 nm Polyscience, Inc. 17152
Water bath VWR 462-7001
Thermostat IKA-Werke GmbH & Co. KG 3164000
Peristaltic pumps Ismatec ISM 833
Bubble traps (glass) UNI-GLAS Laborbedarf
Flow heater UNI-GLAS Laborbedarf
Pressure sensor + Software for analyses MSR Electronics GmbH 145B5
Notebook Hewlett Packard
Miniature gas exchange oxygenator Living Systems Instrumentation LSI-OXR
Tygon Tube (ID: 1.6 mm; OD: 4.8 mm) Ismatec MF0028
Tubes for pumps (PharMed BPT; ID: 1.52 mm) Ismatec SC0744
Blunt cannulae (Ø 0.8 mm) Polymed Medical Center 03.592.81
Blunt cannulae (Ø 1.2 mm) Polymed Medical Center 03.592.90
Blunt cannulae (Ø 1.5 mm) Polymed Medical Center 03.592.94
Blunt cannulae (Ø 1.8 mm) Polymed Medical Center 03.952.82
Parafilm VWR 291-1212
Perfusion chamber with tissue holder (plexiglass) Internal technical department Similar equipment is available from Hemotek Limited, UK
Surgical suture material (PremiCron) B. Braun Medical AG C0026005
Winged Needle Infusion Set (21G Butterfly) Hospira, Inc. ASN 2102
Multidirectional stopcock (Discofix C-3) B. Braun Medical AG 16494C
Surgical scissors B. Braun Medical AG BC304R
Dissecting scissors B. Braun Medical AG BC162R
Needle holder B. Braun Medical AG BM200R
Dissecting forceps B. Braun Medical AG BD215R
Automated blood gas system Radiometer Medical ApS ABL800 FLEX
Multi-mode microplate reader BioTek Synergy HT
Liquid scintillation analyzer GMI, Inc. Packard Tri-Carb 2200
Scintillation tubes 5.5 ml Zinsser Analytic GmbH 3020001
Tissue Homogenizer OMNI, Inc. TH-220
pH meter + electrode VWR 662-2779

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