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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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.

The study of the placental barrier function was intensified with the discovery of the thalidomide induced malformations in the 1960's. For obvious reasons translocation studies with pregnant women cannot be performed. Consequently, various alternative models have been developed ^6,7. The most promising and probably most clinical relevant model is the ex vivo human placental perfusion model developed by Panigel and co-workers ^2,3.

Many women are exposed to different xenobiotics such as drugs or environmental pollutants during their pregnancy ⁸. For some drugs which were already administered regularly during pregnancy, in vivo studies can be performed by comparison of the maternal blood concentration with that in umbilical cord blood. However, generally there is only limited information about the pharmacokinetics and -dynamics in the fetus and the teratogenicity of these substances.

For example opiates like heroin easily cross the placental barrier and can lead to intrauterine growth restriction, preterm delivery or spontaneous abortion ^9,10. So, in case of missing abstinence during pregnancy a replacement therapy with methadone is recommended. The ex vivo human placental perfusion model revealed that the transfer of methadone into the fetal circulation is negligible ¹¹, which correlates well with the calculated cord blood-to-maternal blood concentration ratio after delivery ¹².

Nanotechnology is a growing field especially in medicine. So, beneath the naturally occurring fine (< 2.5 μm in diameter) and ultrafine particles (< 0.1 μm in diameter) in fumes of forest fires, volcano eruptions and in desert dust, exposure to engineered nanomaterials (at least one dimension < 0.1 μm ¹³) is increasing. This raised questions about the toxicological potential of engineered nanomaterials. Although no human hazard could be proved yet, there are principal experimental studies indicating that engineered nanoparticles can cause adverse biological responses leading to toxicological outcomes ¹⁴. Recently, some studies indicated that prenatal exposure to air pollution is linked to a higher respiratory need and airway inflammation in newborns and children ^15,16. In addition, small nanoparticles might be used as drug carriers to specifically treat either the fetus or the mother. Therefore, it becomes evident that extensive studies of distinct xenobiotics or nanomaterials and their ability to cross the placental barrier are required. An actual overview on the current studies on placental permeability to engineered nanomaterials is summarized in Menezes et al. 2011 ¹⁷ and Buerki-Thurnherr et al. 2012 ⁷.

The ex vivo dual recirculating human placental perfusion model provides a controlled and reliable system for studying the placental transport of various endogenous and exogenous compounds ^{3,11,12,18,19} and a wide range of other functions of the placenta like mechanisms responsible for the development of pathological states like preeclampsia ^20-22. In this protocol we focus mainly on the set up, handling and method that allow the study of accumulation, effects and translocation rates of a broad set of xenobiotics or nanoparticles.

Protokół

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 the water bath, the flow heaters and the heating for the perfusion chamber. The temperature should be 37 °C.
3. Warm up the perfusion medium (NCTC-135 tissue culture medium diluted 1:2 with Earle's buffer (6.8 g/L sodium chloride, 0.4 g/L potassium chloride, 0.14 g/L monosodium phosphate, 0.2 g/L magnesium sulfate, 0.2 g/L calcium chloride, 2 g/L glucose) supplemented with glucose (1 g/L), dextran 40 (10 g/L), bovine serum albumin (10 g/L), sodium heparin (2,500 IU/L), amoxicilline (250 mg/L) and sodium bicarbonate (2.2 g/L); pH 7.4) in the water bath.
4. Consecutively rinse the arterial systems of the fetal and maternal circuit with a) 200 ml distilled water, b) 50 ml 1% sodium hydroxide, c) 1% phosphoric acid and d) again 200 ml distilled water (flow rate: 15 - 20 ml/min).
5. Connect the fetal cannula (Ø 1.2 mm; blunt needle should be attached to a modified winged needle infusion set) to the fetal arterial tubing.
6. Rinse the arterial systems of the fetal and maternal circuit with perfusion medium until all tubes contain medium (flow rate: 15-20 ml/min). During this step fill up the bubble traps and remove all bubbles downstream of the trap. Then stop the pumps. It is really important that the afferent arterial tubes are always free of bubbles; otherwise after cannulation especially the fine fetal vessels can rupture.
7. Turn on the gas flow. The maternal circuit is oxygenated with 5% carbon dioxide and 95% synthetic air and the fetal circuit with 5% carbon dioxide and 95% nitrogen.
8. Start the recording of the pressure sensor.

2. Cannulating the Placenta

1. Obtain intact placentae from uncomplicated term pregnancies after primary cesarean section. Written consent has to be given (was obtained in the case of our studies) by the mothers before delivery and the study has to be approved by the local ethics committee (was the case in our studies). First visual control should be done by midwives to assure a healthy and intact placenta.
2. Cannulation of the placenta is a critical step! During perfusion every small disruption in the tissue can lead to a leak between the maternal and fetal circulation. The placenta has to be obtained within 30 min after delivery.
3. Select an intact cotyledon at the marginal zone of the placenta without visible disruptions on the maternal side. At the chorionic plate, tie up both associated branches of the umbilical artery and vein upstream to the later cannulation side (towards the umbilical cord) by using surgical suture material. Make always two knots.
4. Cannulate the fetal artery first. The fetal placental arteries are always smaller and thinner than the veins.
5. Make a suture around the fetal artery, but do not tie it up immediately. Hold the vessel with a forceps, cut the vessel carefully and put the small cannula (Ø 1.2 mm) in the artery. Then tie up the suture (two knots).
6. Proceed with the fetal vein in the same manner but use a bigger cannula (Ø 1.5-1.8 mm; blunt needle should be attached to a modified winged needle infusion set).
7. Turn on the fetal pump (2 ml/min). If there is no visible leak and blood emanates out of the fetal vein cannula, slowly increase the flow up to 4 ml/min. Observe the pressure in the fetal artery, it should not exceed 70 mmHg. If fluid leaks out at the fetal or maternal cannula fix them with another suture.
8. Place the placenta on the tissue holder with the fetal side up and pull the placental membrane and tissue over the spikes. In the end the perfused cotyledon should be in the middle of the hole in the tissue holder.
9. Stabilize the part where only the membrane holds the placenta with a silicone membrane (Ø 1 mm) or alternatively two parafilm pieces.
10. Assemble the complete tissue holder, tighten the screws and cut the overhanging tissue. Please note that the venous and arterial cannulae are not pinched but instead lay in the small channels of the tissue holder.
11. Turn the tissue holder upside down, put it into the perfusion chamber and add the cover. Now, the maternal side should be at the top. Check always if the fetal circuit is still intact and the medium is flowing out of the fetal vein tubing.
12. Turn on the maternal pump (12 ml/min). Introduce the three blunt cannulae (Ø 0.8 mm) at the end of the maternal artery tube into the intervillous space by penetrating the decidual plate. To return the perfusate to the maternal circuit put one tube as venous drain which is also connected with the maternal pump to the lowest position in the upper part of the perfusion chamber.
13. Connect the fetal vein cannula to the fetal vein tube.

3. Executing the Pre- and Experimental Phase of Perfusion

1. To allow the tissue to recover from the ischemic period after delivery and to flush out the blood in the intervillous space, an open pre-phase of 20 min is necessary. That means the maternal and fetal vein are not leading back to the arterial reservoir containing the perfusion medium. Collect the fetal and maternal venous outflow in a bottle and discard it after the pre-phase.
2. To assess the integrity of the perfusion perform another pre-phase of 20 min but in a closed circuit. Use two separate reservoirs with perfusion medium for the fetal and maternal circuit and close the circuits by leading the fetal venous outflow back in the fetal reservoir and the maternal venous outflow back in the maternal reservoir.
3. For the main perfusion experiment prepare two flasks with 120 ml perfusion medium (one for the maternal and one for the fetal reservoir). Add the radiolabeled ¹⁴C-antipyrine (4 nCi/ml; serves as positive control; CAUTION: radioactive substance) and the fluorescently labeled xenobiotic or nanoparticles which one wants to analyze to the maternal reservoir. Mix the maternal perfusate well.
4. Start the experiment by exchanging the pure perfusion medium with the two prepared flasks (fetal and maternal reservoirs). Close the circuits by leading the fetal venous outflow back in the fetal reservoir and the maternal venous outflow back in the maternal reservoir.
5. Continue the perfusion for 6 hr and take samples regularly. Always resuspend the medium in the fetal and maternal reservoir before withdrawal.
6. Control the pressure in the fetal artery (should not exceed 70 mmHg), pH in both circuits (should be in a physiological range 7.2-7.4) and the volume of both reservoirs (fetal volume loss should not exceed 4 ml/hr) during perfusion. If necessary adjust the pH values using either hydrochloric acid or sodium hydroxide.
7. If the volume loss in the fetal reservoir exceeds 4 ml/hr there is a leak in the tissue and one has to stop the perfusion. The success rate of a perfusion for 6 hr without leak is about 15-20%.
8. Stop the perfusion after 6 hr. Turn out the pumps, water bath, flow heaters and gas flow.
9. Remove the placenta from the tissue holder, cut the perfused cotyledon (brighter than the unperfused tissue) and weigh it.
10. Take samples from unperfused (part of the placenta which was cut in the beginning; could be already taken during the pre-phase) and perfused tissue (each about 1 g) and store them at -20 °C until homogenization or in liquid nitrogen for later analysis. Fix another tissue sample in 4% formalin for histopathological evaluation. The samples should include all layers of the placenta.
11. Clean the tubes after perfusion by successively rinsing the arterial systems of the fetal and maternal circuit with a) 200 ml distilled water, b) 50 ml 1% sodium hydroxide, c) 50 ml 1% phosphoric acid and d) again 200 ml distilled water (flow: 15-20 ml/min).

The entire working procedure of the placenta perfusion experiment is depicted in Figure 3.

4. Analyzing the Samples

1. Centrifuge the perfusate samples for 10 min at 800 x g before analysis to remove residual erythrocytes. Take the supernatant for the further analysis. The samples can be left overnight at 4 °C. For the analysis of leptin and hCG production the samples can be stored at -20 °C.
2. To evaluate the permeability of the placenta analyze the ¹⁴C-antipyrine by liquid scintillation. Mix 300 μl of the fetal and maternal samples with 3 ml scintillation cocktail and measure for 5 min in a beta counter.
3. To assess the transfer of the fluorescent nanoparticles or the xenobiotic of interest read the fluorescence at 485 nm excitation and 528 nm emission in a microplate reader (indicated wavelengths are for analysis of the yellow green label which we used for the nanoparticles).
4. To determine the viability of the placental tissue during perfusion measure the glucose consumption and lactate production in the fetal and maternal circuit with an automated blood gas system. Additionally, evaluate the production of the placental hormones human choriongonadotropin (hCG) and leptin in the homogenized tissue samples and the perfusates by enzyme-linked immunosorbent assay (ELISA).

Wyniki

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 ...

Dyskusje

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...

Materiały

Name	Company	Catalog Number	Comments
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 ( 0.8 mm)	Polymed Medical Center	03.592.81
Blunt cannulae ( 1.2 mm)	Polymed Medical Center	03.592.90
Blunt cannulae ( 1.5 mm)	Polymed Medical Center	03.592.94
Blunt cannulae ( 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