JoVE Logo
Faculty Resource Center

Sign In

Summary

Abstract

Introduction

Protocol

Representative Results

Discussion

Acknowledgements

Materials

References

Bioengineering

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

Published: June 18th, 2013

DOI:

10.3791/50401

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.

.css-f1q1l5{display:-webkit-box;display:-webkit-flex;display:-ms-flexbox;display:flex;-webkit-align-items:flex-end;-webkit-box-align:flex-end;-ms-flex-align:flex-end;align-items:flex-end;background-image:linear-gradient(180deg, rgba(255, 255, 255, 0) 0%, rgba(255, 255, 255, 0.8) 40%, rgba(255, 255, 255, 1) 100%);width:100%;height:100%;position:absolute;bottom:0px;left:0px;font-size:var(--chakra-fontSizes-lg);color:#676B82;}

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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 .......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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.......

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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

....

Log in or to access full content. Learn more about your institution’s access to JoVE content here

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
EQUIPMENT
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

  1. Ala-Kokko, T. I., Myllynen, P., Vahakangas, K. Ex vivo perfusion of the human placental cotyledon: implications for anesthetic pharmacology. Int. J. Obstet. Anesth. 9, 26-38 (2000).
  2. Panigel, M., Pascaud, M., Brun, J. L. Radioangiographic study of circulation in the villi and intervillous space of isolated human placental cotyledon kept viable by perfusion. J. Physiol. (Paris). 59, 277 (1967).
  3. Schneider, H., Panigel, M., Dancis, J. Transfer across the perfused human placenta of antipyrine, sodium and leucine. Am. J. Obstet. Gynecol. 114, 822-828 (1972).
  4. Enders, A. C., Blankenship, T. N. Comparative placental structure. Adv. Drug Deliv. Rev. 38, 3-15 (1999).
  5. Takata, K., Hirano, H. Mechanism of glucose transport across the human and rat placental barrier: a review. Microsc. Res. Tech. 38, 145-152 (1997).
  6. Saunders, M. Transplacental transport of nanomaterials. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 1, 671-684 (2009).
  7. Buerki-Thurnherr, T., von Mandach, U., Wick, P. Knocking at the door of the unborn child: engineered nanoparticles at the human placental barrier. Swiss Med. Wkly. 142, w13559 (2012).
  8. Gendron, M. P., Martin, B., Oraichi, D., Berard, A. Health care providers' requests to Teratogen Information Services on medication use during pregnancy and lactation. Eur. J. Clin. Pharmacol. 65, 523-531 (2009).
  9. Burns, L., Mattick, R. P., Lim, K., Wallace, C. Methadone in pregnancy: treatment retention and neonatal outcomes. Addiction. 102, 264-270 (2007).
  10. von Mandach, U. Drug use in pregnancy. Ther. Umsch. 62, 29-35 (2005).
  11. Malek, A., Obrist, C., Wenzinger, S., von Mandach, U. The impact of cocaine and heroin on the placental transfer of methadone. Reprod. Biol. Endocrinol. 7, 61 (2009).
  12. Hutson, J. R., Garcia-Bournissen, F., Davis, A., Koren, G. The human placental perfusion model: a systematic review and development of a model to predict in vivo transfer of therapeutic drugs. Clin. Pharmacol. Ther. 90, 67-76 (2011).
  13. International Organization for Standardization (ISO). Technical Specification (ISO/TS) 27687. Nanotechnologies – Terminology and definitions for nano-objects – Nanoparticles, nanofibre and nanoplate. , (2008).
  14. Pietroiusti, A. Health implications of engineered nanomaterials. Nanoscale. 4, 1231-1247 (2012).
  15. Latzin, P., Roosli, M., Huss, A., Kuehni, C. E., Frey, U. Air pollution during pregnancy and lung function in newborns: a birth cohort study. Eur. Respir. J. 33, 594-603 (2009).
  16. Lacasana, M., Esplugues, A., Ballester, F. Exposure to ambient air pollution and prenatal and early childhood health effects. Eur. J. Epidemiol. 20, 183-199 (2005).
  17. Menezes, V., Malek, A., Keelan, J. A. Nanoparticulate drug delivery in pregnancy: placental passage and fetal exposure. Curr. Pharm. Biotechnol. 12, 731-742 (2011).
  18. Muhlemann, K., Menegus, M. A., Miller, R. K. Cytomegalovirus in the perfused human term placenta in vitro. Placenta. 16, 367-373 (1995).
  19. Wick, P., et al. Barrier capacity of human placenta for nanosized materials. Environ. Health Perspect. 118, 432-436 (2010).
  20. Dancis, J. Why perfuse the human placenta. Contrib Gynecol. Obstet. 13, 1-4 (1985).
  21. May, K., et al. Perfusion of human placenta with hemoglobin introduces preeclampsia-like injuries that are prevented by alpha1-microglobulin. Placenta. 32, 323-332 (2011).
  22. Guller, S., et al. Protein composition of microparticles shed from human placenta during placental perfusion: Potential role in angiogenesis and fibrinolysis in preeclampsia. Placenta. 32, 63-69 (2011).
  23. Challier, J. C. Criteria for evaluating perfusion experiments and presentation of results. Contrib. Gynecol. Obstet. 13, 32-39 (1985).
  24. Kraemer, J., Klein, J., Lubetsky, A., Koren, G. Perfusion studies of glyburide transfer across the human placenta: implications for fetal safety. Am. J. Obstet. Gynecol. 195, 270-274 (2006).
  25. leal, J. K., et al. Modification of fetal plasma amino acid composition by placental amino acid exchangers in vitro. J. Physiol. 582, 871-882 (2007).
  26. athiesen, L., et al. Quality assessment of a placental perfusion protocol. Reprod. Toxicol. 30, 138-146 (2010).
  27. Myllynen, P., et al. Preliminary interlaboratory comparison of the ex vivo dual human placental perfusion system. Reprod Toxicol. 30, 94-102 (2010).
  28. Malek, A., Sager, R., Schneider, H. Maternal-fetal transport of immunoglobulin G and its subclasses during the third trimester of human pregnancy. Am. J. Reprod. Immunol. 32, 8-14 (1994).
  29. Prouillac, C., Lecoeur, S. The role of the placenta in fetal exposure to xenobiotics: importance of membrane transporters and human models for transfer studies. Drug Metab. Dispos. 38, 1623-1635 (2010).
  30. Poulsen, M. S., Rytting, E., Mose, T., Knudsen, L. E. Modeling placental transport: correlation of in vitro BeWo cell permeability and ex vivo human placental perfusion. Toxicol. In Vitro. 23, 1380-1386 (2009).
  31. Mathiesen, L., Rytting, E., Mose, T., Knudsen, L. E. Transport of benzo[alpha]pyrene in the dually perfused human placenta perfusion model: effect of albumin in the perfusion medium. Basic Clin. Pharmacol. Toxicol. 105, 181-187 (2009).

This article has been published

Video Coming Soon

JoVE Logo

Privacy

Terms of Use

Policies

Research

Education

ABOUT JoVE

Copyright © 2024 MyJoVE Corporation. All rights reserved