Background

Traditionally neglected by scientists, the placenta has long been regarded as a temporary organ with basic nutritional functions. Now, it is recognized as a complex and essential organ for mammalian reproduction, as aberrations in its formation and/or function have immediate consequences on pregnancy outcomes, as well as lifelong impacts on the health of the offspring (Malhotra et al., 2019). At term, the placenta has a discoid shape, with one side facing the amniotic cavity (the chorionic plate) and the other in contact with the endometrium (the decidual plate). Its microstructure is formed of villi that could be compared to multiple trees, which come from the branches of the fetal arteries (Turco and Moffett, 2019). Since these villi are directly in contact with the maternal blood, the human placenta is told hemochorial (Foidart et al., 1992). The interface between the maternal blood and the fetal vessels is called the syncytium. This unique layer constitutes the functional unit of the placenta and comes from the fusion of the mononuclear villous cytotrophoblasts (CTBs) to form multinucleated syncytiotrophoblasts (STBs). This process, known as syncytialization, takes place throughout the pregnancy and is critical for the formation and the functions of the placenta (Costa, 2016). On the other hand, the CTBs can also stay mononuclear but acquire an invasive capacity, especially during the first trimester. In this case, they are called extravillous trophoblasts (EVTs) and are crucial to the adaptations of the maternal arteries to pregnancy (Chang et al., 2018). In both cases, the study of CTBs differentiation represents one of the keys to improve our understanding of placental development and related diseases.

There are no perfect experimental models to investigate the human placenta as the differences between mammals are considerable, even in species with hemochorial placentas (e.g., rodents and non-human primates) (Roberts et al., 2016). Additionally, disorders such as preeclampsia, in which the primary defect is a failure in placentation, are found only in humans (Brosens et al., 2011). For these reasons, fundamental research on placental biology was initially based on ex vivo observations of histologic sections (Burton et al., 1999). In the last decades, various in vitro models based on human material were developed and characterized. However, each in vitro system has its benefits and limitations and should be carefully selected based on the trophoblast subtype, the gestational age of interest, and the type of experiment conducted (Colson et al., 2020b). In this context, primary human trophoblasts isolated from normal placenta at term and cultured in plastic cell culture dishes is an easy, cost-effective, and reproducible method to study specific molecular and genetic regulations in the placenta after the first trimester of gestation. This protocol is based on the method originally described by Kliman (Kliman et al., 1986) but was significantly improved by our team. The main advantages of this technique are its simplicity, its efficiency, and its cost. In addition, the method gives reproducible results as we have been able to characterize many independent cultures of several days (up to 12 days) over the last 10 years (Debieve et al., 2013; Depoix et al., 2013, 2016, 2018 and 2020; Colson et al., 2020a). Furthermore, the 2D culture is a flexible method that can be adapted to various experimental conditions (transfection, drug exposure, metabolic study, observations, etc.), allowing mechanistic explorations of cellular processes.

Limitations of this technique are the need for ethical permission, a good liaison with clinical staff, and the innate variability between samples. However, this latter limitation can be avoided by carefully selecting the placentas (similar gestational age, parity, and maternal condition) and repeated experiments. To overcome these difficulties, many research groups have turned to cell lines, including human embryonic stem cell-derived trophoblast cells (Amita et al., 2013), human trophoblast stem cells (Okae et al., 2018), and choriocarcinoma cell lines. Even if the first two cell lines require further characterization, their use is justified when the study is focused on early placentation as first-trimester trophoblasts are difficult to obtain. However, the use of choriocarcinoma cell lines such as JAR, JEG3, or BeWo to study CTB differentiation and functions poses real methodological difficulties. There are growing concerns about the purity, identity, and behavior of these common cell lines (Abbas et al., 2020). For instance, cAMP must be added to the culture medium to induce BeWo differentiation, while primary CTBs spontaneously fuse in vitro. Finally, genome-wide DNA methylation studies showed significant variability in comparison with primary cells, which is likely to contribute to differential expression profiles (Apps et al., 2011; Novakovic et al., 2011). Therefore, it becomes difficult to draw definitive conclusions when using such cell lines, which highlights the importance of validating findings by using primary trophoblasts.

Another alternative to primary human CTB cell culture is the use of placental explants from human placentas. Their culture is very easy and has the main advantage of keeping the CTBs in their complex tissue environment. It allows the comparison between placentas from normal pregnancies and pathological placentas (preeclampsia or fetal growth restriction) (Orendi et al., 2011). However, studies have shown that this technique impairs trophoblast viability and induces an unnatural oxidative stress (Di Santo et al., 2003; Goncalves et al., 2016). Since then, the use of explants in the study of placental biology must be restricted to comparisons with pregnancy-related diseases and must be limited in time. Finally, 3D cell culture models of human trophoblast cells that closely mimic the villous placenta were recently developed (Haider et al., 2018; Turco et al., 2018). These organoids, composed of a layer of CTBs surrounding a core of STBs, can also be differentiated into EVTs. These models undoubtedly represent a breakthrough in the understanding of placental biology but require specific technical skills and a consequent budget. For these reasons, we are convinced that primary human term CTB cultures present undeniable advantages that still justify their place in the placental field.