Ex Vivo Placental Explant Flow Culture - Mimicking the Dynamic Conditions In Utero

We are trying to discover where some pregnant women develop preeclampsia and others don't. This pregnancy-specific syndrome causes hypertension and proteinuria of the mother and may cause death of mother and child. With our translational research, we hope to identify the placental causes leading to the maternal symptoms.

Recently, single-cell RNA sequencing approaches have been used to identify differences between healthy and preeclamptic placentas. Now comes the time to link this data with the original tissues. This is why we're using spatial histology and institute sequencing to identify changes on the single-cell level.

The individual complexity of the geometric structure of the placenta presents a challenge for determining the exact flow velocity and shear stress for a given placental villus. With the use of this flow culture system, the importance of shear force on the biological functions of placental tissues can be addressed and investigated. To begin, place the excised pieces of placental samples in PBS.

Discard the chorionic plate, the maternal decidua, and any visible infarcts from the specimen. Dissect the remaining tissue specimen into villus explants with a cross-sectional diameter of about 0.5 centimeters. Transfer the explants into a Petri dish filled with fresh PBS.

With a pair of forceps, shake each explant in the PBS to remove all blood. Under a sterile hood, use luer locks to connect five chambers to the reservoir bottle. Invert the chambers upside down and remove the bottom.

Now use forceps to place the metal plates centrally in the top parts of the chambers with the pins pointing upwards. Fill half of the chambers with one milliliter of prewarmed culture medium. Add an additional 20 milliliters of the medium into the reservoir.

With forceps, carefully place the villus explant onto the needles of the metal plate in the chamber. Place four explants into a single chamber. Reattach the bottom of the chambers to close it.

Next, connect the pump tubing to the pump to connect the flow circuit with the peristaltic pump inside the bioreactor. Fix it on the fourth stage. Under the Pumps menu, choose the Manual mode.

Adjust the pump speed to one milliliter per minute and click on RUN to start pumping. Hold the chambers at an angle during filling to ensure complete filling. When the filling process is finished, invert the chamber again.

Confirm that the chambers are secure and close both lids of the bioreactor. Once the tissue incubation is complete, click ABORT to stop the pump. At a time, open two bioreactor lids and one flow chamber.

Use forceps to carefully remove the explants from the metal plate for further analysis. Immunohistochemically stained explants showed a well-structured and organized visual presentation of the cytoskeleton in fresh-and flow-cultured tissue. Over time, microfilament aggregation was increasingly observed in static explants, signifying a degradation of the cytoskeletal structure.

The diminishing tissue integrity over cultivation time was confirmed by hematoxylin and eosin staining. Fresh tissues showed a dense and tightly packed stroma. After 48 hours of flow culture, partially detached fragments of the syncytiotrophoblast were seen.

The tissue integrity was inadequately preserved already after 24 hours in a static culture condition, which deteriorated further after 48 hours. Endothelial cell staining showed distinctively organized cells in the fresh tissue. The morphological integrity was widely maintained even after 48 hours in the flow cultures.

The static explants, however, exhibited partial collapse even after 24 hours, which deteriorated further after 48 hours.