Name: Ex vivo Culture of Human Placental Explants for the Study of Viral Transmission Across the Maternal-Fetal Interface (Video) | JoVE Journal
Uploaded: 2025-12-30T14:00:00.000+00:00
Duration: 5 min 22 s
Description: Learn ex vivo culture of human placental explants at an air-liquid interface to study viral transmission across the maternal-fetal interface. This method enables investigation of gestation-dependent viral replication using cytomegalovirus and Zika virus models.

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5 minutes

ChM and LG doctoral theses were funded by the French Ministry of Education and Research (MESR). This work received government funding managed by ANRS MIE under France 2030, reference number &#34;ANRS-24-PEPRMIE-0004&#34; in the frame of the PEPR VERTICAL project. CEM also received financial support from the D&#233;fi Cl&#233; Risques Infectieux et Vecteurs Occitanie (RIVOC ZIKEV-sf) and Agence de la Biom&#233;decine (24AMP009). Our team received institutional grants from INSERM, CNRS, and Toulouse University. We warmly thank the medical and paramedical staff of the gynecology/obstetrics department at Paule de Viguier Hospital, who allowed us to have access to the samples, as well as the patients who agreed to participate in the study. We also thank the Germeth&#232;que for hosting the biological collection. We warmly thank the following people for the generation of some illustrative data: Mathilde Bergamelli, Ma&#239;lys Mouysset, Marta Freixas Teres, Jean-Michel Mansuy, J&#233;r&#244;me Ausseil, Yannick Simonin, as well as the whole ViNeDys team for daily discussions about our projects. We finally thank Ga&#235;tan Ligat for careful reading of the manuscript and Emma Bordes for help in experiments. Figure 1 was created in&#160;https://BioRender.com; Creative Commons Attribution License (CC-BY).

Experiments must be conducted according to the local ethical guidelines. For this study, the biological resource center Germeth&#232;que Toulouse (BB-0033-00081) obtained the written informed consent from each patient for the use of samples (CPP.2.15.27). The steering committee gave its approval for the realization of the study on February 5th, 2019. The hosting request made to Germeth&#232;que bears the number 20190201, and its contract is referenced under the number 19 155C. The biological resource center has a declaration DC-2014-2202 and an authorization AC-2015-2350. In accordance with confidentiality policies, the only information collected is the date of the sample collection (week of pregnancy). Appropriate safety conditions must be followed for all steps of this protocol (BSL2 laboratory and personal protective equipment (PPE)). Figure 1 illustrates an overview of the protocol. The reagents and the equipment used are listed in the Table of Materials.
1. Material preparation
NOTE: This step aims to prepare all the material necessary for the exploitation of a sample.
<ol>
	<li>Autoclave the forceps and scissors.</li>
	<li>Prepare the media:
	<ol>
		<li>Medium without FBS (Fetal Bovine Serum): Dulbecco&#39;s Modified Eagle Medium (DMEM) + 100 U/mL penicillin/streptomycin (P/S) + 100 mg/mL normocin + 2.5 mg/mL amphotericin B.</li>
		<li>Complete medium: DMEM + 10% FBS + 100 U/mL penicillin/streptomycin (P/S) + 100 mg/mL normocin + 2.5 mg/mL amphotericin B</li>
	</ol>
	</li>
</ol>
2. Collection and visual examination of the sample
NOTE: These steps aim to assess the usability of the sample. For first-trimester placentas, a simple visual observation of the sample is not sufficient; the following steps must be carried out. At the end of these steps, if the quality of the placenta is satisfactory, continue to the next steps. All blood waste must be considered infectious; the pipettes and utensils used must be decontaminated after use.
<ol>
	<li>Prepare the workbench by placing two sterile 15 cm dishes. Fill one dish with one-third PBS, and leave the other empty. Prepare a waste container. 
	NOTE: Dishes can be put on an alcohol-soaked paper towel, which will provide contrast to better identify tissue structures.</li>
	<li>Transfer the entire sample into the empty dish. 
	NOTE: If the sample contains a lot of blood, aspirate the excess with a 25 mL pipette and discard it before the transfer.</li>
	<li>Using sterile forceps, isolate all placental structures and place them into the 15 cm dish containing the PBS. 
	NOTE: It is easier to start by removing easily recognizable structures that are not of interest with forceps (e.g., cervical mucus (long, viscous, elastic filaments), blood clots (dark red masses)). Aspiration of excess blood allows tissue structures to be seen more clearly. If in doubt about a structure, place it in PBS and shake it gently to clean it (successive grip and release) and observe its morphology. At first glance, the placenta has a sponge-like appearance. The umbilical cord is fairly easy to spot in the sample (fairly large white vessels), and the placenta will be at the end. The villi of the placenta appear as coral-like branches, light in color (light pink/white). If the placenta has been damaged during surgery, the villi will be distributed throughout the sample, and the sorting process will be long and tedious. The sample will be unusable if too much blood has coagulated and trapped the tissue structures, or if the placenta is too damaged, meaning that no tissue structure is intact enough to obtain 1 mm3 pieces.</li>
	<li>For a full-term placenta, skip steps 1 to 3 and place the placenta in a sterile 30 cm x 20 cm surgical tray.</li>
</ol>
3. Preparation and dissection of the placenta
NOTE: These steps describe how to dissect the placenta into 1 mm3 explants that will be incubated overnight before being cultured at the air/liquid interface.
<ol>
	<li>Prepare another sterile 15 cm dish with PBS. Using sterile forceps, transfer the placental structures, gently shaking them to remove as much blood as possible. 
	NOTE: To save plastic, use the (sterile) lid of these dishes as a container and add PBS (adjust the volume).</li>
	<li>Using two sterile forceps, tear the placental villi into small pieces (i.e., explants) of approximately 1 mm3. 
	NOTE: Size is estimated visually; when an explant is lifted, it will appear as a small droplet. Explants that are too small will be unusable; explants that are too large can be reduced in size the next day when placing them on the sponges.</li>
	<li>To facilitate the counting, group explants in batches of 10 in the dish containing PBS. Make as many batches as needed for the experiment. 
	NOTE: Be careful not to move the dishes once they are placed in PBS; the batches are not stable and will shift with movement, making them difficult to count later. Plan on having more explants than needed for the experiment (the explants can disintegrate, and small pieces cannot be used). In general, count between 3 and 6 sponges per condition (3 or 6 x 10 explants).</li>
	<li>Once the desired quantity of explants is obtained, group them into one well per experimental condition using sterile forceps and add the appropriate amount of medium, following the instructions: (1) Fewer than 30 explants: in a 24-well plate, 1 mL medium; (2) 30 to 60 explants: in a 12-well plate, 1.5 mL medium; (3) More than 60 explants: in a 6-well plate, 2 mL medium. 
	NOTE: If no infection is planned for the histoculture, add complete medium. If a viral infection is planned, add medium without FBS for both the infected and non-infected conditions. Medium can be put in the well before transferring the explants. Then go to step 4 below.</li>
	<li>Place the plate in an incubator at 37 &#176;C, 5% CO2, overnight.</li>
	<li>For a full-term placenta, use pieces of tissue collected in the thickness of the sample, in regions facing the fetal side of central cotyledons, without retaining the surface layer. Place them in a 15 cm dish containing PBS. Then proceed as with first-trimester placentas. Prepare explants of 1 mm3 by cutting with scissors instead of using forceps, starting at step 3.2.</li>
</ol>
4. Infection
NOTE: If there are any infections or treatments to be carried out, perform them before placing the plate in the incubator overnight at the previous step.
<ol>
	<li>Instead of adding medium to recover the explants overnight (step 3.4 above), add the virus preparation at the desired multiplicity of infection (MOI). Complete with medium without FBS if needed to reach the total volume of medium indicated in step 3.4. 
	NOTE: Since the MOI initially refers to individual cells, different virus concentrations should be tested to determine the appropriate one.</li>
	<li>Gently agitate manually with a tip or forceps so that all explants in the well are in contact with the infected medium. Do not flush the explants with a pipette to perform this step.</li>
	<li>Place the plate in the incubator overnight or for the desired duration (37 &#176;C, 5% CO2).</li>
</ol>
5. Hydration of gelatin sponges
NOTE: One quarter of a sponge is used for nine explants. Hydration of gelatin sponges has to be done the same day as the dissection. This way, the sponges will be ready for explant installation the next day.
<ol>
	<li>In a sterile 6-well plate, put 3 mL of medium without FBS in each well that will contain a sponge.</li>
	<li>Gently hold the sponge with sterile forceps, without applying excessive pressure to avoid deforming it. Using a pair of sterile scissors, cut the sponge into four equal parts. 
	NOTE: For ease of use, pre-cut the sponge a few mm in the middle, lengthwise, then cut the first quarter.</li>
	<li>Place one sponge per well, counting nine explants per sponge. 
	NOTE: Cut directly above the desired well so that the sponge falls inside. Be careful, sponges are very light and volatile; the presence of medium allows them to stay in the well.</li>
	<li>Replace the unused sponges in their packaging and reseal them tightly for further use.</li>
	<li>Place the 6-well plate containing the sponges overnight in the incubator, 37 &#176;C, 5% CO2.</li>
</ol>
6. Washing of explants and installation on gelatin sponges
<ol>
	<li>The next day, renew the culture medium (3 mL of medium per well, complete medium or medium without FBS, depending on whether the infection step has been performed or not).</li>
	<li>In a sterile 6-well plate, put sterile PBS into each well to fill three-quarters.</li>
	<li>Place a sterile strainer in the first well and, using sterile forceps, transfer all the explants from a given condition to the strainer.</li>
	<li>Gently mix with the forceps and shake the strainer for about 10 s so that all the explants are in contact with the PBS.</li>
	<li>Manually move the strainer to the second well of the plate and repeat step 4. Continue moving the strainer until the 6th well. 
	NOTE: The PBS turns red when the explants are first introduced into the strainer. At the last wash, the PBS should remain colorless. If not, repeat the washes in a new sterile 6-well plate containing PBS.</li>
	<li>Using forceps, place the explants one by one on the sponge (9 explants per sponge). 
	NOTE: Explants are often heterogeneous in size despite the initial dissection. It is more practical to place the largest explants in the center and the four corners of the sponge, with smaller ones in between. The explants should not touch each other. It is helpful to work with two forceps; explants that are too large are divided (torn) into smaller ones.</li>
	<li>Put the plate back in the incubator at 37 &#176;C, 5% CO2.</li>
	<li>Repeat steps 2-7 for each explant processing condition.</li>
</ol>
7. Culture medium renewal and monitoring
<ol>
	<li>Every 3-4 days, up to the end of the culture, collect the culture supernatant from the various wells and put the entire volume in a sterile 50 mL tube, renew it with 3 mL of complete medium preheated to 37 &#176;C. This volume should allow for partial submergence of the collagen sponge, maintaining the tissue at the air/liquid interface. 
	NOTE: The clarified culture supernatant can be used to monitor different parameters (e.g., explant viability, viral release, etc). Set aside a 500 &#181;L aliquot and store it at -20 &#176;C or -80 &#176;C until further use. If explants have fallen from the sponges and are found in the medium during the culture, replace them on the sponge either with sterile forceps or with a 1000 tip (aspiration with the 1000 &#181;L pipette).</li>
	<li>On the last day of the culture (between 14-21 days), using sterile forceps, collect the explants and place them individually or in groups in cryotubes, previously labeled and weighed empty. Weigh the tubes again to obtain the tissue mass. 
	NOTE: Depending on the intended use, it is useful to store the explants in different formats, for example, 3 tubes containing 1 explant, 3 tubes containing 3 explants, and the remaining explants by groups of 10. It is also useful to weigh them individually and record their mass for tubes containing 1 and 3 explants.</li>
	<li>Set aside the desired explants for histology (3 are sufficient for routine procedures). Add 500 &#181;L of formalin for 24 h before dehydration, paraffin embedding, and cryosection.</li>
	<li>Flash-freeze the remaining explants by putting the cryotubes directly into liquid nitrogen in a Dewar container. 
	CAUTION: Wear appropriate PPE (gloves, goggles, coat), as there is a risk of splashing. Use airtight cryotubes suitable for freezing in liquid nitrogen (if not, there is a risk of explosion).</li>
	<li>Recover the cryotubes using a Chinese-style metal strainer and store them at -80 &#176;C.</li>
</ol>

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<li>Coste Mazeau, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Potential+of+anti-CMV+immunoglobulin+Cytotect+CP+in+vitro+and+ex+vivo+in+a+first-trimester+placenta+model%5BTitle%5D)+AND+%22Microorganisms%22%5BJournal%5D)">Potential of anti-CMV immunoglobulin Cytotect CP in vitro and ex vivo in a first-trimester placenta model.</a> Microorganisms. 10 (4), 694(2022).</li>
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The issue of vertical transmission of viruses and their replication in the placenta is a major public health concern1,20. To address this issue and conduct studies that will lead to a better understanding of the interactions between viruses and the placenta, various models exist, each with its own advantages and limitations11. In this article, the detailed protocol for a human placental histoculture model using placental explants cultured at the air-liquid interface is presented.
The first requirement is to be able to obtain human samples, at different stages of pregnancy if needed, in collaboration with medical teams from a nearby gynecology/obstetrics department, since there must be a short time between the collection of the placenta at the hospital and its preparation in the laboratory. Ethical considerations must also be taken into account. The collection of these samples is subject to strict regulations that vary by country. Researchers must complete the required regulatory procedures with ethics committees and obtain written informed consent from patients.
The critical part of the protocol lies mainly in assessing the quality of the placenta upon receipt and before its preparation, particularly for first-trimester placentas. The trophoblastic part must be clearly visible, the villi must not disintegrate during dissection, and there must not be too much blood clot in the sample. Once the dissection has been performed and the explants installed on the sponges, it is also necessary to monitor the samples for possible fungal or bacterial contamination. This is quickly visible as a white discoloration appearing on the tissue and a medium that may become cloudy, sometimes accompanied by a characteristic odor. In such cases, the entire culture and culture media must be discarded, and the incubator and culture hoods decontaminated.
Although very close to physiology, this model has several limitations. The first lies in its limited manipulability. The cellular organization within the tissue is complex, making it difficult to manipulate and/or monitor events in a given cell type. It also has inherent variability due to differences between human donors, which limits experimental reproducibility but increases significance.
Using this model, researchers can evaluate the placenta&#39;s permissiveness to various viruses at different stages of pregnancy and the consequences of viral infection on placental functions over a long period of time. This has been done with HCMV17,21,22,23, ZIKV3,24,25, USUV19, and SARS-CoV-226, for example. This model also allows to evaluate the tissue response to infection, including the production of cytokines, pro-inflammatory mediators, and placental extracellular vesicles22,27,28. Finally, placental histocultures are a precious tool for evaluating novel therapeutic strategies. They have been used to measure the efficacy of antiviral chemical compounds29 or hyperimmune globulins on HCMV replication30 or to evaluate protection against ZIKV infection following vaccination27.

Congenital viral infections are a major public health concern. Between 0.5% and 1% of births worldwide are affected by human cytomegalovirus (HCMV) infection1. The recent emergence of several flaviviruses, such as Zika virus (ZIKV) and Oropouche virus (OROV), has also highlighted the growing threat that viral infections pose to pregnant women and their fetuses2,3,4. In this context, there is a need for tools to investigate vertical transmission, particularly the key step of viral replication in the placenta during different stages of pregnancy.
The placenta is a key organ during pregnancy, enabling the exchange of nutrients, gases, and waste products5. It also acts as a barrier against infection by most pathogens, but, as mentioned above, it can also be targeted by certain viruses that are able to replicate in this organ6. The placenta is composed of different cell types that are organized into repetitive basic units called placental villi (composed of cytotrophoblasts, fused syncytiotrophoblasts forming a continuous external layer, mesenchymal cells, endothelial cells, and fetal macrophages, also known as Hofbauer cells), which are bathed in maternal blood and also anchor themselves to the maternal uterine matrix (also known as the decidua) and invade it via the extravillous cytotrophoblasts, allowing the remodeling of the maternal spiral artery5. All of these cell types organize themselves to form a very precise architecture, which, by maintaining itself, enables maternal-fetal communication.
It is important to note that placental structure and function change substantially across gestation, which can impact viral tropism and pathogenesis7. Early pregnancy placentas are characterized by a relatively thick syncytiotrophoblast layer, abundant cytotrophoblasts, and a less developed villous tree with fewer fetal capillaries. By term, the villous tree becomes more extensive, the syncytiotrophoblast thinner, stromal and vascular networks densify, and immune cell populations such as Hofbauer cells shift in abundance and activation8. These developmental transitions are paralleled by major transcriptomic and proteomic changes between first- and third-trimester placentas9,10. Together, these stage-specific structural, molecular, and immunological shifts shape placental susceptibility to pathogens, which can vary across gestational age.
Among the various models used to study the interaction between viruses and the placenta, which include placental cell lines, primary cells, and placental organoids11,12, one placental histoculture model, consisting of the culture of fresh placental tissue obtained from pregnant women at different stages of pregnancy, is presented in this manuscript. A major advantage of this histoculture model, in addition to its evident physiological added-value compared to in vitro models, is that it preserves the organ&#39;s cytoarchitecture and the interaction between different placental cell types, for an extended period of time by culturing tissue explants at the air/liquid interface on gelatin sponges. Such histocultures permit nutrient and gas diffusion from a liquid phase while keeping the tissue surface exposed to air, preserving differentiated tissue architecture and matrix interaction compared with fully submerged explants. This configuration allows long-term viability and functional readouts compared to the culture in standard plates with or without extracellular matrix coating or transwell, which generally describes tissue culture maintenance of two to three days13,14,15. Air/liquid histocultures have been shown to better maintain the integrity, differentiation, and cytokine secretion of various human tissues, including lymphoid, cervical, and skin explants for extended periods of time16. For placental models, it has been reported to preserve villous structure and sustain viral replication for up to three weeks17,18, which is crucial when studying viruses like HCMV, which take a long time to replicate and disseminate into the tissue. Hence, this configuration provides enhanced physiological relevance and longevity compared with submerged explants.
In this article, the steps involved in preparing placental histocultures from first-trimester or full-term placentas to study viral infections and assess therapeutic tools are presented. After dissecting the organ, the tissue explants are infected and cultured at the air/liquid interface on gelatin sponges. Tissue viability is monitored during culture. Various virological parameters can then be examined, including viral titers in the supernatant and/or in the tissue, as well as cytokine secretion.
The protocol described below can be performed using placentas at different stages of pregnancy. For a so-called &#34;full-term&#34; placenta, the sample is obtained at approximately 9 months&#39; term, i.e., around 39 &#177; 1 weeks of pregnancy. To avoid labor-associated inflammation, which may influence tissue responsiveness to viral infection, non-labored, cesarean section deliveries should be preferred over labored or vaginally delivered placenta. The sample is collected in a sterile plastic container and then stored at +4 &#176;C for a maximum of 16 h before use. Only patients with no sign of illness, for the mother and the baby, are selected.
For a first-trimester placenta, the sample is collected in the context of a voluntary pregnancy termination. The product of aspiration of the patient&#39;s uterine contents is collected in a vacuum-sealed box and is placed at +4 &#176;C. It can be stored for a maximum of 16 h before being used (usually, the sample is handled the same day it is collected). The gestational ages range from 6-12 weeks of pregnancy, the terms of the placentas used are generally between 7 and 11 weeks of pregnancy for greater repeatability (before 7 weeks of pregnancy, the placenta is often too small to obtain a usable quantity).

<table><tbody><tr><td>Amphotericin B (250 &amp;micro;g/mL)</td><td>Gibco</td><td>15290026</td><td>Explant culture</td></tr><tr><td>Cryotubes</td><td>Greiner</td><td>123280</td><td>To store tissues and aliquots of supernatants</td></tr><tr><td>Dish 15 cm</td><td>Sarstedt</td><td>83.3903</td><td>For dissection and manipulation of explants</td></tr><tr><td>DMEM [1X] 4.5 g/L D-Glucose with Glutamax I, Pyr. de Sodium</td><td>Invitrogen</td><td>31966047</td><td>Explant culture</td></tr><tr><td>Foetal Bovine Serum</td><td>Sigma</td><td>F7524</td><td>Explant culture, do not use during the infection step</td></tr><tr><td>Normocin (50 mg/mL)</td><td>Invivogen</td><td>ant-nr-2</td><td>Explant culture</td></tr><tr><td>Penicillin-Streptomycin solution, liquid, (10,000 units/mL)</td><td>Invitrogen</td><td>15140122</td><td>Explant culture</td></tr><tr><td>Phosphate Buffer Saline (PBS)</td><td>Sigma</td><td>D8537</td><td>For dissection and washing steps</td></tr><tr><td>Stainless steel dissecting forceps, length 11 cm</td><td>Dutscher</td><td>5086</td><td>For dissection and manipulation of explants</td></tr><tr><td>Straight scissors with pointed blades</td><td>Dutscher</td><td>711193</td><td>To perform dissection and cut sponges</td></tr><tr><td>Strainers 100 &amp;micro;m</td><td>Falcon</td><td>352360</td><td>For washing step</td></tr><tr><td>SURGIFOAM Absorbable Gelatin&lt;br/&gt; Sponges by Ethicon Size (12-7), 12/CS</td><td>Ethicon</td><td>ETH-1972</td><td>Distributor: Medex Supply</td></tr></tbody></table>

ex vivo culture of human placental explants for the study of viral transmission across the maternal-fetal interface

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

The placenta plays a critical role during pregnancy, enabling maternal-fetal exchange while serving as a barrier against pathogens. However, some viruses, including human cytomegalovirus (HCMV) or Zika virus (ZIKV), can cross this barrier and cause congenital infections, leading to severe fetal outcomes such as growth restriction, impairment of brain development, or fetal demise. Understanding how viruses infect and disseminate through the placenta is therefore essential. To investigate viral vertical transmission in a physiologically relevant context, an ex vivo model using human placental explants cultured at the air-liquid interface on gelatin sponges can be used and offers several advantages. Unlike conventional in vitro systems limited to single-cell types, this model preserves the multicellular structure and function of the placental tissue for up to three weeks. This model is compatible with a wide range of upstream experimental applications (histological analyses, RT-qPCR, TUNEL assay, quantification of secreted factors, etc.), making it a robust and accessible platform to study viral replication, immune responses, and placental barrier function under near-physiological conditions.

This protocol describes the preparation of fresh human placental tissue explants cultured at an air-liquid interface, from first- or third-trimester placenta, for the study of viral infections. The timeline of the experiment is shown in Figure 2A. One of the first critical steps, especially for the 1st-trimester placenta, is to examine by eye the tissue integrity after its collection before proceeding to the dissection of trophoblastic villi, taking out non-trophoblastic structures, notably maternal decidua, and avoiding the presence of blood clot. Representative pictures of placental samples before, during, and after dissection and culture on gelatin sponges are shown in Figure 2B,C.
This protocol allows the maintenance of the tissue viability for up to two weeks, and up to three weeks if needed, which is a duration appropriate to study the dissemination of viruses into the explants and their immune response. Several experiments can be done to check the tissue health and integrity over the duration of the culture. When the culture medium is renewed (every 3-4 days), a sample of the supernatant can be kept to follow the presence of the β-human chorionic gonadotropin (β-HCG), indicating its secretion by trophoblastic cells. A decrease in b-HCG secretion during the culture is generally observed and does not preclude the proper viability and integrity of the tissue18 (Figure 3A). Tissue cytoarchitecture can also be assessed at the end of the culture (or with an explant collected earlier if needed) by submitting one or several explants to immunohistochemistry against placental alkaline phosphatase (PLAP), cytokeratin 7 (CK7), both expressed by the syncytiotrophoblast layer, as well as vimentin (VIM), expressed by the mesenchymal cells18 (Figure 3B). Viability of the tissue can also be assessed by performing a TUNEL assay, which allows the detection of cleaved DNA in dying cells. No signal is expected if the placenta is viable, in contrast to a positive control realized upon a DNase I pretreatment of the tissue, as shown in Figure 3C18,19.
Placental histocultures can be used for assessing the impact of viral infection on placental function and immune response. To this aim, an overnight step of infection with viral preparation, followed by extensive washing of the explants to eliminate the viral inoculum, is needed before their installation on gelatin sponges. During the 14-21 days of culture, the efficiency of infection can be monitored in several ways, as exemplified in Figure 4, which illustrates representative results that have been obtained upon infection by HCMV, ZIKV, or Usutu virus (USUV). Histoculture supernatant can be collected throughout the duration of the experiment to measure viral replication and release of viral particles, by performing RT-qPCR, as shown for HCMV18 and ZIKV (Figure 4A,C). Analyses can also be done directly on placental tissue at the end of the culture to evaluate the expression of viral genes and proteins, by performing either immunohistochemistry18 (as shown for HCMV in Figure 4B) or RT-qPCR to detect the expression of viral transcripts19 (as shown for USUV in Figure 4D). These data also illustrate the inherent variability of the results obtained with this model, due to the intrinsic interindividual variability for human samples. For example, the HCMV titer determined on histoculture supernatant varied from 1.05 x 104 and 1.53 x 107 copies/mL depending on the placental sample, with most of the experiments falling into the 105 copies/mL range18 (Figure 4A). During ZIKV infection, the efficiency of the viral replication also varies between the donors, with one explant (PLA#2) that seems not permissive for ZIKV replication (Figure 4C). In such cases, the viral inoculum is rapidly cleared due to the washes performed after incubation of the explants, prior to their installation on the sponges. Consequently, the viral titers decline rapidly over time in non-permissive donors. This variability is also observed for the expression of USUV transcripts for the different donor, in both first and third trimester explants19 (Figure 4D). Finally, it has to be noted that placental explants may have a different permissiveness depending on the gestational term for some viruses. In this line, the infection of third-trimester placental explants is nearly impossible with this protocol for HCMV. To circumvent this limitation, sponges can be put in a culture well containing infected permissive cells (such as MRC-5) which will continuously produce infectious particles and allow the infection of the explants17. However, this procedure could then prevent the possibility to carry out the analysis of supernatant during the time of co-culture, since both producer cells and placental explants will contribute to the secretion of viral particles and soluble mediators.
<img alt="Placental explant process: diagram of collection, dissection, infection, hydration, installation." class="xfigimg" src="/files/ftp_upload/69525/69525fig1.jpg" data-altupdatedat="1774324804000" data-alt="Figure 1"> 
Figure 1: Schematic representation of the different steps of placenta dissection into explants, infection, and installation on gelatin sponges. <a href="https://www.jove.com/files/ftp_upload/69525/69525fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Placental cell culture process; timeline, Petri dishes, tissue incubation; step-by-step experiment." class="xfigimg" src="/files/ftp_upload/69525/69525fig2.jpg" data-altupdatedat="1774324804000" data-alt="Figure 2"> 
Figure 2: Illustration of different steps of the protocol. (A) Timeline of the placental histoculture protocol. (B) Representative pictures of the different steps of the dissection of a first-trimester placenta. The left dish contains the unprocessed tissue, the middle dish contains a trophoblastic part of the placenta being currently dissected, and the right dish contains several (at least 50) tissue explants after dissection. (C) Placental explants maintained in culture on gelatin sponges in a 6-well plate. The medium of the middle well is being collected. The right picture shows an individual sponge with nine explants at its surface (CNRS image/ViNeDys Lab). <a href="https://www.jove.com/files/ftp_upload/69525/69525fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="β-HCG graph and TUNEL assay results; immunohistochemistry for PLAP, CK7, VIM in trimester tissues." class="xfigimg" src="/files/ftp_upload/69525/69525fig3.jpg" data-altupdatedat="1774324804000" data-alt="Figure 3"> 
Figure 3: Monitoring of placental viability and integrity. (A) Monitoring of β-HCG secretion in histoculture supernatant from first-trimester placentas. β-HCG was measured in the culture medium between days 3-5 (measure 1) and days 10-13 (measure 2). ** p = 0.0011 by paired t-test (n = 12 independent histocultures). This figure is modified from Bergamelli et al.18. (B) Representative images of immunohistochemistry and hematoxylin staining carried out on 1st-trimester (Top) and 3rd-trimester (Bottom) placental explants collected after 15 days of culture, against placental alkaline phosphatase (PLAP), cytokeratin 7 (CK7), and vimentin (VIM). Scale bar = 100 mm. (C) Representative images of TUNEL assay (right panels) of 1st-trimester (Top) and 3rd-trimester (Bottom) done on placental explants collected after 15 days of culture. These images are modified from Martin et al.19. <a href="https://www.jove.com/files/ftp_upload/69525/69525fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="HCMV ZIKV titer graphs, microscopy image; viral RNA analysis, 1st and 3rd trimester results." class="xfigimg" src="/files/ftp_upload/69525/69525fig4v2.jpg" data-altupdatedat="1774324804000" data-alt="Figure 4"> 
Figure 4: Monitoring of the infection of placental explants by HCMV (A,B), ZIKV (C), or USUV (D). (A) Quantification by qPCR of the HCMV genome in the histoculture supernatant of first-trimester placental explants collected at 7-13 days post-infection. (B) Representative image of immunohistochemistry staining performed on first-trimester placental explant after 15 days of culture upon infection by HCMV (NI: non-infected), performed against HCMV Immediate Early antigen, and counterstained with hematoxylin. Scale bar = 50 mm. These images were modified from Bergamelli et al.18. (C) Quantification by RT-qPCR of the ZIKV RNAs in the histoculture supernatant of first-trimester placental explant collected at day 1, 2, and 5 post-infection (dpi). Three independent placental histocultures (#1 to #3) have been infected with either ZIKV (solid line) or UV-treated ZIKV (UV; dotted line). (D) Viral RNA was extracted from placental explants (1st or 3rd trimester) upon infection by USUV or by UV-inactivated virus (USUV UV), for six independent experiments (NI: non-infected), and quantified by RT-qPCR using the ΔCT method (normalized by actin). The ΔCT values are represented by a double gradient color map (blue: high ΔCT = no amplification of viral RNA; red: low ΔCT = amplification of viral RNA; and crossed gray: no data). A one-way ANOVA statistical test was performed followed by Tukey's multiple comparison test (ns, non-significant; *, p &lt; 0.05; ***, p &lt; 0.0005; ****, p &lt; 0.0001). These images are modified from Martin et al.19.<a href="https://www.jove.com/files/ftp_upload/69525/69525fig4largev2.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

This protocol describes the preparation of human placental tissue explants cultured at an air-liquid interface for the study of viral infections.

The placenta plays a critical role during pregnancy, enabling maternal-fetal exchange while serving as a barrier against pathogens. However, some viruses, ...

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.

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Immunology and Infection

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

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