Ex vivo Culture of Human Placental Explants for the Study of Viral Transmission Across the Maternal-Fetal Interface

Our research focuses on how viral replications takes place in human placental tissues to decipher the mechanisms of vertical transmission. Our protocol addresses the lack of physiologically relevant long-term human placental models to study gestation-dependent viral replication and transmission. To begin, use sterile forceps to transfer the placental structures, gently shaking them to remove as much blood as possible.

With two pairs of sterile forceps, tear the placental villi into small pieces of approximately one cubic millimeter. To facilitate counting, group explants in batches of 10 in a dish containing PBS. Make as many batches as needed for the experiment.

Once the desired quantity of explants is obtained, add an appropriate amount of medium to a sterile 12-well plate and group the explants into one well per experimental condition using sterile forceps. Add the virus preparation at the desired multiplicity of infection. Then gently agitate manually with a tip or forceps, so that all explants in the well are in contact with the infected medium.

Next, put three milliliters of medium without fetal bovine serum into each well of a sterile six-well plate. Gently hold a gelatin sponge with sterile forceps without applying excessive pressure to avoid deforming it. With a pair of sterile scissors, cut the sponge.

Place one sponge into each well, counting nine explants per sponge. Then place the six-well plate with the sponges overnight in the incubator at 37 degrees Celsius and 5%carbon dioxide. The next day, put sterile PBS into each well of a new sterile six-well plate to fill 3/4 of the volume.

Place a sterile strainer in the first well, and using sterile forceps, transfer all the explants from a given condition to the strainer. Gently mix with the forceps and shake the strainer for about 10 seconds, so that all the explants are in contact with the PBS. Now, manually move the strainer to the second well of the plate and repeat the washing procedure.

Continue moving the strainer until the sixth well. After the last wash, use forceps to place the explants one by one on the sponge, positioning nine explants per sponge. Placental tissue integrity was visually assessed before dissection to exclude non-trophoblastic structures and blood clots prior to explant preparation.

Placental explants were successfully cultured on gelatin sponges at the air-liquid interface after dissection. Beta-human chorionic gonadotropin secretion decreased during the culture period, but did not preclude the proper viability and integrity of the tissue. Placental cytoarchitecture was preserved during culture with placental alkaline phosphatase, cytokeratin 7, and vimentin expression detected in both first and third trimester explants.

No apoptotic signal was detected in untreated placental explants in contrast to the DNase I treated positive control. Human cytomegalovirus replication was detected in histoculture supernatants with viral titers varying between placental donors. Human cytomegalovirus proteins were detected in infected placental explants by immunohistochemistry.

Zika virus replication in placental explants varied between donors, with some samples showing limited permissiveness. Use of viral transcripts were detected in both first and third trimester placental explants with donor-dependent variability. Our protocol preserves placental tissue architecture and cell interactions, allowing long-term study of viral replication and tissue responses.

The current challenges include donor viability, limited manipulability of complex tissue, short sample availability, and risk of microbial contaminations. Future research will focus on studying emerging viruses, their impact on pregnant women, and testing antiviral strategy to prevent vertical transmission.