# Human placental models for studying viral infections

Charlène Martin, Mathilde Bergamelli, Hélène Martin, Mélinda Bénard,
Charlotte Tscherning, Cécile E Malnou

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Charlène Martin, Mathilde Bergamelli, Hélène Martin, Mélinda Bénard, Charlotte Tscherning, et al. Human placental models for studying viral infections. Current Opinion in Virology, 2025, 71, pp.101454. 10.1016/j.coviro.2025.101454. hal-05523057

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Review

# Human placental models for studying viral infections

Charlène Martin<fnref n="1" />, Mathilde Bergamelli<fnref n="2" />,<fnref n="3" />, Hélène Martin<fnref n="1" />, Mélinda Bénard<fnref n="1" />,<fnref n="4" />, Charlotte Tscherning<fnref n="1" />,<fnref n="5" /> and Cécile E Malnou<fnref n="1" />

<figure><img src="image_2.png" /></figure>

Viral infections during pregnancy represent a major threat to maternal, fetal, and neonatal health outcome, with a high risk of vertical transmission. It is therefore crucial to understand the mechanisms underlying the interaction between viruses and placenta, which ensures communication between maternal and fetal compartments throughout pregnancy. Human placental models, both *in vitro* and *ex vivo*, enable to dissect in detail these interactions. By studying in detail viral entry, replication, and immune responses within the placenta, they represent ideal tools for analyzing the effects of various viruses on pregnancy outcomes. In addition, these models serve as platforms for evaluating diagnostic and therapeutic approaches to protect pregnant women and their babies from viral infections. This review examines recent advances, the main advantages and limitations of different human placental models and discusses their potential to improve our understanding of virus-placenta interactions, thereby contributing to improved maternal and fetal health.

## Addresses

Corresponding author: Malnou, Cécile E (cecile.malnou@univ-tlse3.fr)

### Current Opinion in Virology 2025, 71:101454

This review comes from a themed issue on **Ex-vivo models for virus research**

Edited by **Cyrille Mathieu** and **Bertrand Pain**

For complete overview about the section, refer "Ex-vivo models for virus research (2024)"

Available online 13 March 2025

https://doi.org/10.1016/j.coviro.2025.101454

1879-6257/© 2025 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

## Introduction

In a world where congenital human cytomegalovirus (HCMV) infection affects 0.7% of births [1], where the rubella virus still causes up to 100 000 cases of congenital rubella syndrome per year despite the existence of a vaccine (WHO, 2024) and where many other viruses such as Zika virus (ZIKV) are associated with severe anomalies during pregnancy, all interests are turning to the importance of the placenta. This organ plays a pivotal role, particularly in gas and nutrient exchanges, maternal immune regulation, and defense against pathogens [2]. During viral congenital infections, it may act as a barrier or a gateway for viruses to reach the fetus and can also allow the transfer of antiviral treatments. Therefore, gaining a deeper understanding of the intricate interactions between viruses and the placenta is essential for enhancing maternal and fetal health.

While immortalized laboratory cell lines have many limitations and *in vivo* animal models are far from the physiological development of the human placenta, primary cells, organoids, and *ex vivo* human placental models constitute precious tools to examine its various physiological and pathological aspects, allowing in-depth study of the interactions between viruses and this key organ. A major feature of these placental models lies in their complex cell composition and structure, which allow a comprehensive study of the different cell types of the placenta, including cytrophoblasts, syncytiotrophoblasts, and endothelial cells among others. This enables an accurate assessment of virus's ability to infect these various cell types and alter essential placental functions such as invasion, differentiation, or immune barrier.

The development of these models and their use in virology began with the study of placental tropism and replication of HCMV and HIV-1 [3,4]. Since 2015, alongside emergence of arboviruses outbreaks, they have also been of great interest for studying impact of ZIKV and other flaviviruses on pregnancy [5,6]. More recently, they have also been used to analyze the impact of SARS-CoV-2 replication on placenta [7]. In addition to deciphering viral infection mechanisms and the consequences of viral infections on placenta biology, placental models are also used to assess the efficacy of

<footnote n="1">¹Institut Toulousain des Maladies Infectieuses et Inflammatoires (Infinity), Université de Toulouse, INSERM, CNRS, UPS, Toulouse, France</footnote>

<footnote n="2">²Division of Obstetrics and Gynaecology, Department of Clinical Science, Intervention and Technology (CLINTEC), Karolinska Institute, Stockholm, Sweden</footnote>

<footnote n="3">³Department of Gynecology and Reproductive Medicine, Karolinska University Hospital, Stockholm, Sweden</footnote>

<footnote n="4">⁴CHU Toulouse, Hôpital des Enfants, Service de Néonatalogie, Toulouse, France</footnote>

<footnote n="5">⁵Division of Neonatology, Oslo University Hospital, Oslo, Norway</footnote>
potential antiviral therapies and predict their ability to cross the placental barrier to potentially protect the fetus [8,9].

In this review, we will examine different human placental models and evaluate their advantages and limitations in the context of viral infections. Primary trophoblast culture, placental explant culture, placental organoids, and dual lobe perfusion technologies will be discussed, as well as their application in the assessment of viral pathogenesis and antiviral therapy.

## Primary trophoblast culture

The trophoblast is a cell layer that surrounds the blastocyst around day 5 of development. It later differentiates into two cell types: cytotrophoblasts, which form the inner layer, and syncytiotrophoblasts, which form the outer layer and facilitate the invasion of the maternal endometrium. Primary trophoblast culture consists of *in vitro* culture of purified trophoblast cells, freshly prepared from human placental tissue. This simplified reductionistic model allows the study of trophoblast biology and functions with controlled parameters to investigate various aspects, including proliferation, differentiation, invasion, or innate immune response (Table 1). However, neither the interaction with other placental cell types nor the tissue structure can be studied by this approach.

The methodological principle of primary trophoblast culture involves the isolation of trophoblast cells from fresh placenta, at any term of pregnancy. It has been developed by Kliman et al in 1986, followed by many steps of technological development [10–12]. The isolation procedure typically begins with sequential enzymatic digestions of fresh placental tissue with trypsin and DNase to release trophoblast cells, followed by density centrifugation methods. The technique can be optimized by immunoselection methods to obtain a relatively pure population. The isolated trophoblasts are then seeded onto culture dishes coated with extracellular matrix proteins, such as collagen or fibronectin, and cultured in a specialized medium supplemented with growth factors and hormones that support

trophoblast proliferation and differentiation [13]. Cultures can be maintained for around 1 month under controlled conditions [14] and may be immortalized [15].

Primary trophoblasts have been used to study their susceptibility to various viral infections and the subsequent effects on their function. For example, it has been shown that HCMV infection of trophoblasts suppresses their ability to differentiate [16,17] and that trophoblast stem cells can restrict HCMV infection [18]. The assessment of their permissiveness by ZIKV constituted the first clue of the transplacental route for ZIKV vertical transmission [19,20]. Other studies also investigated how primary trophoblasts can restrict replication of several viruses [21,22].

## Placental explant cultures

Human placental explants from early to late pregnancy are relevant tools to study viral infections due to their unique characteristics along each developmental stage (Table 2). First-trimester explants offer insight into initial stages of placental development and susceptibility to viral infection during critical periods of embryogenesis. On the other hand, third-trimester explants provide a window to the mature placental response to viruses, including its ability to mount immune responses. Hence, these models allow for a comprehensive understanding of whether the placenta differentially responds to viral infections and how these infections impact its functions throughout the entire duration of pregnancy, thanks to the preserved cytoarchitecture and complex cell composition.

The methodological principle of human placental explant culture involves the isolation and maintenance of intact tissue fragments in a controlled environment, preserving the structural integrity and cellular composition of the placenta (Figure 1a) [23,24]. Typically, placental tissue is processed to generate explants, which are cultured on specialized substrates (such as gelatin sponges for culture at air-liquid interface or Transwell inserts), ensuring optimal viability and functionality up to 3 weeks. By maintaining tissue cytoarchitecture and cellular interactions, placental explant culture enables

<table><caption>**Table 1**</caption><thead><tr><th colspan="2">Advantages and limitations of primary trophoblast culture.</th></tr><tr><th>Advantages</th><th>Limitations</th></tr></thead><tbody><tr><td><strong>Homogeneous population:</strong> Relatively pure population of cells, allowing focused study of a specific cell type, with controlled culture conditions.</td><td><strong>Loss of tissue architecture:</strong> Lack of 3D structure and cytoarchitecture present in placental tissue, limiting the ability to accurately recapitulate physiology.</td></tr><tr><td><strong>Reproducibility:</strong> High control of experimental conditions, facilitating the investigation of molecular mechanisms.</td><td><strong>Limited heterogeneity:</strong> Loss of the diversity of cell types, leading to oversimplification of complex biological processes.</td></tr><tr><td><strong>Proliferative potential:</strong> Can proliferate and be passaged in cell culture, allowing a generation of sufficient cell amount for downstream assays and experiments.</td><td><strong>Senescence and differentiation:</strong> May undergo senescence and/or differentiate over time, affecting phenotype and functionality.</td></tr></tbody></table>
<table><caption>**Table 2**

**Advantages and limitations of placental explant cultures.**</caption><thead><tr><td>Advantages</td><td>Limitations</td></tr></thead><tbody><tr><td><p><strong>Tissue architecture preservation:</strong> Maintenance of native architecture with multiple cell types and study of complex cellular interactions within the tissue.</p><p><strong>Functional assays:</strong> Assessment of functional parameters such as nutrient transport, hormone secretion, and immune responses, providing insights into placental physiology.</p><p><strong>Ex vivo viability:</strong> Remain viable and functional for 3 weeks, allowing middle-term studies.</p></td><td><p><strong>Heterogeneity:</strong> Inherent variability due to differences in donor characteristics, limiting experimental reproducibility, but increasing significance.</p><p><strong>Limited manipulability:</strong> Structurally complex, challenging to manipulate individual cell populations or control cellular organization within the tissue.</p><p><strong>Ethical considerations:</strong> ethical issues regarding donor consent and tissue procurement, potentially limiting accessibility and scalability.</p></td></tr></tbody></table>

the investigation of complex processes, including viral dissemination, replication, and immune responses, providing valuable insights into the pathogenesis of viral infections.

Using this model, characterization of placenta permis-
siveness for HCMV throughout pregnancy has been
studied [25], along with its impact on cytokines and
proinflammatory mediators production [26,27], as well as
on placental extracellular vesicles composition and

function [24]. Recent studies have expanded the use of
placental explant cultures to investigate the effects of
arboviruses on placental tissues. ZIKV, along with other
neurotropic viruses such as West Nile, Powassan, and
Usutu viruses, can replicate in placental explants,
leading to fetal demise in murine models [6,28]. ZIKV-
infected explants show pro-inflammatory responses and
apoptosis [29], with enhanced infection in the presence
of Dengue virus antibodies [30,31]. Studies on Hepatitis
E [32], SARS-CoV-2 [7], and Rift Valley fever virus [33]

<figure><img src="image_3.png" /><figcaption>**Figure 1**</figcaption></figure>

Schematic representation of ex vivo placental models to study viral infections. (a) Histocultures: The placenta is dissected in small explants that are then cultured on gelatin sponges at the air-liquid interface. (b) Dual lobe perfusion: A placental lobule is isolated and cannulated at both maternal and fetal sides. Created via Biorender.com.
<table><caption>**Table 3**</caption><thead><tr><th colspan="2">Advantages and limitations of placental organoids.</th></tr><tr><th>Advantages</th><th>Limitations</th></tr></thead><tbody><tr><td><p><strong>Simple:</strong> Simplified and standardized model, facilitating experimental manipulation and control over culture conditions.</p><p><strong>Spatial organization:</strong> Mimicking the three-dimensional architecture of the placenta, facilitating intercellular communication.</p><p><strong>High-throughput screening:</strong> Easily generated in large numbers in multiwell plates, enabling high-throughput screening of drugs or various experimental conditions.</p></td><td><p><strong>Limited complexity:</strong> Lack of the full complexity of native tissue, limiting their ability to recapitulate certain physiological processes.</p><p><strong>Standardization challenges:</strong> Variability in size, shape, and cellular composition between batches, experimental variability which complicates data interpretation.</p><p><strong>Technological expertise:</strong> Requires specialized expertise, potentially limiting accessibility.</p></td></tr></tbody></table>

further highlight the model utility in exploring viral impacts at the maternal-fetal interface.

Placental explants also represent useful platforms for evaluating novel therapeutic strategies. They have been used to measure immune protection against ZIKV infection upon vaccination or novel therapeutics [34], as well as evaluating the effect of hyperimmune globulins on HCMV replication [35].

## Placental organoids

Placental organoids are three-dimensional cell culture models that mimic the microarchitecture and cellular interactions of the placenta *in vitro* (Table 3). Their generation by Turco et al in 2018 represented a groundbreaking advancement in the field of reproductive biology [36,37]. Their structural organization closely resembles the villous placenta with a layer of cytotrophoblasts that differentiate into syncytiotrophoblasts [38]. They also retain migration and invasion abilities.

The methodological principle behind placental organoids involves the aggregation of placental cells into spherical structures, typically using techniques such as hanging drop culture or suspension culture [39]. These organoids self-assemble over time, forming multicellular aggregates that recapitulate spatial organization and some physiological functions of the placenta more accurately than traditional monolayer cultures [40]. Placental organoids offer several advantages, including enhanced cell-cell communication, increased cell viability, and above all the ability to model complex tissue

structures, such as the syncytiotrophoblast layer [38]. Additionally, organoids can be maintained under controlled conditions for several months, facilitating experimental reproducibility and scalability.

Since their development, placental organoids have primarily been utilized to study interactions between the placenta and emerging viruses like ZIKV [41] by showing that ZIKV inhibits syncytialization [42] and to investigate the effects of antiviral drugs [43]. Research on placental organoids has also explored their susceptibility to SARS-CoV-2 [44]. Additionally, these organoids facilitate the study of the innate immune response against viruses such as ZIKV and HCMV at the maternal-fetal interface [45,46].

## Placental dual lobe perfusion

Dual lobe perfusion represents an advanced *ex vivo* experimental approach used with unique features [47–49]. It was initially developed in 1967 by Panigel et al [50] and further improved by Schneider et al [51]. In this technique, the use of a placental lobule — with an intact villous tree — obtained from a consenting donor enables investigations on the uptake, retention, metabolism, and transfer of substances and pathogens across the placental barrier (Table 4).

The methodology typically involves the following steps. The placenta is collected shortly after delivery and transported to the laboratory under controlled conditions. Upon arrival, a placental lobule is cannulated at both maternal and fetal sides (Figure 1b). Cannulation allows for the perfusion of the lobe through the maternal

<table><caption>**Table 4**</caption><thead><tr><th colspan="2">Advantages and limitations of placental dual lobe perfusion.</th></tr><tr><th>Advantages</th><th>Limitations</th></tr></thead><tbody><tr><td><p><strong>Precise control:</strong> Control of the experimental conditions, allowing for standardized experiments and reliable data collection.</p><p><strong>Isolation of variables:</strong> Study of specific variables in a controlled manner without confounding factors from the maternal circulation.</p><p><strong>Real-time observation:</strong> Blood flow dynamics, nutrient transfer, and barrier function for example.</p></td><td><p><strong>Short experimental duration:</strong> Experiments conducted over relatively short durations due to the limited viability outside the body.</p><p><strong>Technical challenges:</strong> Specialized equipment and expertise needed.</p><p><strong>Tissue degradation:</strong> Limiting the duration of experiments and potentially affecting the reliability of results.</p></td></tr></tbody></table>
<table><caption>**Table 5**

Applications of placental models in viral infection studies.</caption><thead><tr><th></th><th>Primary trophoblast culture</th><th>Placental explants cultures</th><th>Placental organoids</th><th>Placental dual lobe perfusion</th></tr></thead><tbody><tr><td>Characterization of viral tropism and replication dynamics of viruses</td><td>CVB [21]<br>HCMV [16–18,21]<br>HSV-1 [21]<br>PV [21]<br>VSV [21]<br>VV [21]<br>ZIKV [19,20]</td><td>CHIKV [28]<br>HEV [32]<br>HCMV [25]<br>MAYV [28]<br>POWV [28]<br>RVFV [33]<br>SCV2 [7]<br>USUV [6]<br>WNV [28]<br>ZIKV [30,31]</td><td>SCV2 [41,44]<br>ZIKV [41]</td><td></td></tr><tr><td>Impact of the viral infection on the placenta</td><td></td><td>HCMV [24]<br>HEV [32]</td><td>ZIKV [42]</td><td></td></tr><tr><td>Viral transmission</td><td></td><td></td><td></td><td>ZIKV [52,53]</td></tr><tr><td>Placental response to viral infection</td><td></td><td>HEV [32]<br>HCMV [25–27]<br>ZIKV [29]<br>HCMV [35]<br>ZIKV [34]</td><td>HCMV [45]<br>ZIKV [46]</td><td></td></tr><tr><td>Evaluation of therapeutic strategies</td><td></td><td></td><td>ZIKV [43]</td><td>HIV [9,54,56,57]</td></tr></tbody></table>

CHIKV: chikungunya virus; CVB: coxsackievirus B3; HCMV: human cytomegalovirus; HEV: hepatitis E virus; HIV: human immunodeficiency virus; HSV-1: herpes simplex virus-1; MAYV: Mayaro virus; POWV: Powassan virus; PV: poliovirus; RVFV: Rift valley fever virus; SCV2: Sars-CoV-2; USUV: Usutu virus; VSV: vesicular stomatitis virus; VV: vaccinia virus; WNV: West Nile virus.

or fetal side of the placenta depending on the experimental design. The perfusion system consists of pumps, reservoirs, and monitoring devices. Once the perfusion system is set up and stabilized, substances of interest, such as viral preparations or drugs, are added to the perfusion medium to assess their transport across the placenta. Sampling ports allow for the collection of perfusate from both the maternal and fetal sides at regular intervals.

This system, with its unique ability to allow the passage of substances through the placental barrier, enables the examination of several key aspects of viral transmission and dissemination. For instance, it has been used to study the transplacental crossing of ZIKV and the impact of cross-reactive dengue virus antibodies on this process [52,53].

Additionally, it serves as an ideal model for examining the pharmacokinetics and placental transfer of antiviral drugs, as demonstrated with HIV antiretroviral therapies [9,54–57]. Despite its power, this model remains however underutilized in virology, largely due to its significant limitation of short viability, which ranges from a few hours to a maximum of 2 days, making it challenging to investigate long-term effects or chronic infections [47,58]. It is, moreover, limited to the study of term placenta.

## Use of placental models in the context of viral infection

The use of these different placental models in the context of viral infection encompasses a multifaceted approach

aimed at understanding various aspects of the interaction between viruses and human placenta throughout pregnancy (Table 5). Initially, research focused on characterizing the tropism and replication dynamics of viruses within placental tissues, shedding light on their ability to infect and propagate within this organ. Studies also often explore how viral infections impact essential placental functions, such as proliferation, invasion, barrier integrity, and metabolism, elucidating some mechanisms underlying pregnancy complications associated with viral exposure. Furthermore, investigations delve into the routes of viral transmission across the placenta, deciphering the pathways through which viruses gain access to the fetal compartment. The immune response elicited by the placenta against various viruses is also a subject of research, aiming to understand how the placenta orchestrates defense mechanisms to protect the developing embryo and fetus. Finally, placental models serve also as precious tools for screening for toxicity, efficacy, and ability to cross the placental barrier of antiviral agents, offering a platform to evaluate the potential of therapeutic interventions in mitigating viral infections and their complications during pregnancy. Overall, the use of placental models provides a comprehensive framework for dissecting the intricate interplay between viruses and the placenta, offering crucial insights for the development of preventive strategies and therapeutic interventions in the context of congenital infections.

## Conclusion

Primary cells, organoids, and *ex vivo* placental models represent a major advance in our ability to study virus–placenta interactions and understand the consequences
of viral infections during human pregnancy. Their increasing
use in research on infectious diseases and emerging viruses
offers great prospects for the development of prevention
and treatment strategies to protect maternal and fetal health.
However, despite their promising potential, placental
models still present challenges. The complexity of cellular
organization and interactions in the placenta requires so-
phisticated experimental approaches and in-depth char-
acterization of the models used. Moreover, the
interindividual variability of placental samples can compli-
cate the standardization of experimental protocols, even
though it represents a rich resource. Finally, an ethical
challenge lies in obtaining fresh placental tissue, particularly
in early and mid-gestation, due to the important considera-
tions surrounding the status of embryos and fetuses.
Addressing these challenges will be crucial for maximizing
the potential of human *ex vivo* placental models in advan-
cing our knowledge and developing effective preventive
and therapeutic strategies in the field of maternal-fetal
health. Such proactive developmental strategies, which
lessen the consequences of viral infections in pregnant
women and neonates, while increasing understanding of the
disorder, will be of profound significance in global health.
Studies on pregnant women are few, as there is a default
position with exclusion of this vulnerable population in
many studies [59]. Therefore, placental models have real
potential to bridge these gaps and alleviate suffering and
save lives.

Funding sources

ChM doctoral thesis is funded by the Ministry of
Education and Research (MESR), France and her work
is supported by the Villa M Foundation, France; MB
postdoctoral position is funded by Wenner-Gren
Foundation (grant no. UPD2022-0108), USA and
Horizon 2020 innovation (ERIN) grant (no. EU952516)
EU. CEM received financial support from the Agence
Nationale de la Recherche Corofet grant (ANR-21-
CE14-0030), France, Défi Clé Cell Based Biotherapies
Occitanie (CeBBOc EXOBARRIER), France, Défi Clé
Risques Infectieux et Vecteurs Occitanie (RIVOC
ZIKEV-sf), France, and Agence de la Biomédecine
(24AMP009), France. Our team received institutional
grants from Inserm, France, CNRS, France, and
Toulouse III University, France.

Data Availability

No data were used for the research described in the art-
ticle.

Declaration of Competing Interest

The authors declare that they have no known competing
financial interests or personal relationships that could
have appeared to influence the work reported in this
paper.

Acknowledgements

We first warmly thank the women and the medical/paramedical staff who contribute daily to medical research on pregnancy and viral infections. We also thank all our colleagues who contribute to our studies, particularly the ViNedys team. We apologize to the teams whose work was not cited here due to space limitation.

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