The overall goal of this procedure is to study the transport of nanoparticles or xenobiotics across the placenta using the ex vivo human placental profusion model. This is accomplished by first cannulating, one fetal artery, and one fetal vein of the placenta immediately after delivery. In the second step, the placenta is fixed in the perfusion chamber.
Blunt cannula are then introduced into the intervillous space on the maternal side, and a single tube is installed to return the venous drain. Next, the whole system is rinsed with perfusion media in an open circulation to flush out the remaining blood and to allow the tissue to recover from the ischemic phase after delivery. In the final step, the two circuits are closed and the fluorescently labeled nanoparticles or the xeno biotic of interest are added to the maternal circulation.
Ultimately, the samples of the fetal and maternal circulation obtained during the six hours of perfusion can be analyzed for the concentration of the nanoparticles or xeno biotic of interest by measuring the fluorescence. The main advantage of this technique over existing methods like animal experiments or cell culture models, is that placental transport studies can be done in a complete organ and directly on human tissue, which is especially important for the placenta as the anatomy of the tissue very strongly between different species. After setting up the profusion system consisting of a water bath, a profusion chamber, two columns for oxygenation, two peristaltic pumps, two bubble traps, two flow heaters, and one pressure sensor.
Connect these components with tubing sections composed of polyvinyl chloride according to this scheme. Then turn on the water bath, the flow heaters, and the heating for the perfusion chamber. As the perfusion media is pre warming in the water bath consecutively, rinse the arterial systems of the fetal and maternal circuits at a flow rate of 15 to 20 milliliters per minute.
Then connect the fetal cannula to the fetal arterial tubing and rinse the arterial systems of the fetal and maternal circuits with prewarm perfusion media until all the tubes contain media. While the tubes are filling, fill the bubble traps, remove all the bubbles downstream of the trap, and then stop the pumps. Now turn on the gas flow and start the recording of the pressure sensor.
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. So work carefully After obtaining intact placenta from uncomplicated term pregnancies.
After primary cesarean section, select an intact co leadin at the marginal zone of the placenta without visible disruptions on the maternal side. Find the same co leadin on the fetal side. At the chorionic plate, tie up both branches of the umbilical artery and vein, which supply the chosen co leadin upstream of the later cannulation site.
Begin by placing a suture around the fetal artery at the chorionic plate. Then holding the vessel with forceps. Cut the artery carefully and put the small cannula in the artery.
Tie the suture with two knots using a bigger cannula. Proceed with the fetal vein in the same manner. Then turn on the fetal pump and slowly increase the flow up to four milliliters per minute.
Blood should emanate outta the fetal vein cannula, and the pressure in the fetal artery should not exceed 70 millimeters mercury. Next, place the placenta on the tissue holder with the fetal side up and pull the placental membrane and tissue over the spikes. The perfused co leadin should be in the middle of the hole in the tissue holder.
Then using a silicone membrane, stabilize the tissue where only the membrane holds the placenta. Now assemble the complete tissue holder. Be certain that the venous and arterial cannula are not pinched, but instead, lay in the small channels of the tissue holder.
Then tighten the screws and trim any overhanging tissue. The placental tissue holder is now fully assembled. Then turn the tissue holder upside down.
Place it into the perfusion chamber and cover the chamber. Confirm that the fetal circuit is still intact and that the media is flowing out of the fetal vein tubing. Now turn on the maternal pump.
Then introduce the three blunt cannula at the end of the maternal artery tube into the intervillous space by penetrating the decidual plate. To return the Perfuse eight to the maternal circuit, place a tube connected with the maternal pump in the lowest position in the upper part of the perfusion chamber to act as a venous drain. Then connect the fetal vein cannula to the fetal vein tube to allow the tissue to recover from the ischemic period after delivery, and to flush out the blood in the intervillous space.
Flush the maternal and fetal vessels with perfusion media for 20 minutes, collecting the fetal and maternal venous outflow in a bottle. To assess the integrity of the perfusion, perform another pre-phase of 20 minutes in a closed circuit using two separate reservoirs with perfusion media for the fetal and maternal circuits. Close the circuits by leading the fetal venous outflow back into the fetal reservoir and the maternal venous outflow back into the maternal reservoir.
For the main perfusion experiment, prepare two flasks with 120 milliliters, each of perfusion media for the maternal reservoir and the fetal reservoir. Add the radio labeled anti pyrene and the desired fluorescently labeled xeno biotic or nanoparticles to the flask for the maternal reservoir and mix the maternal perfu eight. Well finally start the experiment by exchanging the pure perfusion media with the two prepared flasks.
The volume of the fetal and maternal reservoir should remain equal during the six hours of perfusion. A fetal volume loss greater than four milliliters per hour indicates a leak and the perfusion has to be stopped. The success rate of a perfusion for six hours without leak is about 15 to 20%In this first graph, the perfusion profiles of small 80 nanometer polystyrene particles, which were transported across the placenta compared to bigger 500 nanometer polystyrene particles that were not transferred to the fetal compartment are shown for polystyrene nanoparticles.
The placental transfer is size dependent after three hours of placenta perfusion already 20 to 30%of the initially added 80 nanometer polystyrene particles were transferred from the maternal to the fetal circuit, while the 500 nanometer polystyrene particles did not appear in the fetal circuit, even after six hours of perfusion. Anti pyrene is a small lipophilic molecule that is distributed over the placental barrier via passive diffusion and serves as a control for the integrity of the circuits. After four to six hours of perfusion equilibrium between the fetal and maternal anti pyre concentration should be built to assess and compare the placental transport rate of xenobiotics.
The fetal to maternal drug concentration ratio is usually displayed here. A characteristic perfusion profile of the radio labeled anti pyne is depicted through the analysis of placental hormone production as well as glucose consumption and lactate production. The viability and functionality of the placental tissue during the perfusion can be monitored.
After watching this video, you should have a good understanding of how to perform an ex vivo human placental perfusion, which is a valid and reliable method to predict the in vivo passage of various endogenous and exogenous substances.
