**1. Hemochorial Placentation and Fluid Shear Stress**

Human gestation involves so-called hemochorial placentation, which means that maternal blood is in direct contact with the fetal part of the placenta—the chorion frondosum, consisting of placental chorionic villi. However, before perfusion of the placenta with maternal blood, and thus hemochorial placentation, is fully established, a number of fundamental processes occur. Three to four days after fertilization, the morula stage is defined by the occurrence of a totipotent cell mass consisting of approximately sixteen cells. Still in the fallopian tube, but only one day later, cells of the morula differentiate into an inner and an outer cell mass, referred to as the embryoblast and the trophoblast, respectively. Entrance of the blastocyst into the uterine cavity is followed by apposition and adhesion of the blastocyst with its embryonic pole to the endometrial epithelium, enabling implantation of the embryo into the maternal endometrium and subsequent initiation of placentation. As soon as adhesion of the blastocyst is established, trophoblasts located at the embryonic pole (now equivalent with the "implantation pole") start to fuse to form a multinucleated syncytium, referred to as the syncytiotrophoblast. At that very early stage of embryo implantation, the syncytiotrophoblast is the

cell type that enables penetration of the endometrial epithelium and the underlying stroma, which now is referred to as decidua basalis. Once the blastocyst has completely infiltrated the decidual stroma, the syncytiotrophoblast rapidly increases in size by continuing proliferation and fusion of underlying mononucleated cytotrophoblasts. Shortly thereafter, primary placental villi, composed of a cytotrophoblast core and an overlying syncytiotrophoblast layer arise. At the distal regions of these primary villi, cytotrophoblasts breach the syncytiotrophoblast, differentiate into an invasive phenotype, and invade as so-called extravillous trophoblasts the decidual interstitium up to the first third of the myometrium. During migration, extravillous trophoblast subpopulations encounter and invade several luminal structures, including uterine spiral arteries, and veins (endovascular trophoblasts), glands (endoglandular trophoblasts), and to a minor extent uterine lymphatic vessels (endolymphatic trophoblasts) [1–3]. This way, arteries, veins, and glands are connected to the intervillous space to guarantee successful placentation. However, before uteroplacental blood flow is completely established, extravillous trophoblasts accumulate and form cellular plugs that largely obstruct maternal arterial blood flow into the intervillous space until the end of the first trimester of pregnancy. At gestational week six to seven, these trophoblast plugs appear loosely cohesive with clear capillary-sized channels, enabling constant microvascular flux into the intervillous space [4]. Thus, a distinct functional relevance can be attributed to trophoblast invasion into spiral arteries, which results in the remarkable remodeling of vessels, including depletion of smooth muscle cells and loss of the elastic lamina in their walls. The consequence thereof is that opening of spiral arteries into the intervillous space dilate and resemble flaccid conduits, enabling reduction of the velocity of incoming maternal blood and thereby preventing damage to delicate villous trees [5].

With hemochorial placentation, and thus establishment of maternal blood flow, the perfused fetal chorionic tissue has to deal with fluid shear stress, which is produced by plasma and hemodynamic forces across uteroplacental endothelial cells and trophoblast subtypes throughout gestation. Based on a simplified model, fluid shear stress in blood vessels is quantified by the dimension of the inner diameter of the vessel, velocity of flow, and dynamic viscosity, resulting in the force per unit area (dyn/cm<sup>2</sup> = 0.1 Pa) [6]. However, the pulsatile maternal blood flow, the dynamic viscosity of maternal blood, and the micro-anatomical architecture of uterine blood vessels and structure of placental villous trees complicate appraisal of the in vivo fluid shear stress in human utero-placental circulation [7]. Thus, fluidic flow at the uteroplacental interface can be laminar and/or turbulent, resulting in variations in intraluminal forces (Figure 1).

Cells are able to sense fluid shear stress through mechanotransduction, which activates multiple downstream signaling pathways. In addition to endothelial cells, mechanosensing has recently been reported for many other cell types, including trophoblast subtypes that are exposed to fluidic flow. A variety of proteins, receptors, and transmembrane channels are suggested to act as mechansosensors [7,8]. Besides G-protein coupled receptors, integrins, and ion channels, the cytoskeleton of the cell may be involved in mechanosensing. Accordingly, fluidic flow may deform the cellular surface, leading to transduction of forces to cytoskeletal filaments through membrane-spanning proteins, focal adhesion proteins, and glycocalyx components, such as heparin sulfate, chondroitin sulfate, and hyaluronic acid moieties. This interconnection of cellular mechanosensors and cytoskeletal components has led to the concept of a cellular mechanosome complex [8].

During human reproduction, even the preimplantation embryo, including the morula and later on the early blastocyst, is considered to be subject to fluidic flow forces generated by peristalsis of the fallopian tube. However, at this early phase of conception, the zona pellucida — a thick glycoprotein layer, which surrounds the oocyte to allow only species-specific fertilization and persists up to the early blastocyst stage — is suggested to protect the early embryo from harmful mechanical forces [9]. Only with the onset of intervillous perfusion, hemodynamic forces affect the phenotype and physiology of extravillous trophoblasts in invaded uteroplacental spiral arteries as well as villous trophoblasts covering placental villi. Failure of adaptions to this fluid shear stress is suggested to contribute to

pregnancy disorders, including fetal growth restriction, which manifests upon impaired spiral artery remodeling, high vascular resistance, and placental hypoperfusion [7]. *Int. J. Mol. Sci.* **2020**, *21*, x FOR PEER REVIEW 3 of 13

**Figure 1.** Potential route of maternal blood flow into the intervillous space during first trimester. During early placental development, maternal blood flow in maternal uterine spiral arteries is obstructed by extravillous trophoblast plugs. However, parts of maternal blood (red arrows) can pass through narrow intertrophoblastic gaps when trophoblast plugs begin to dissolve during first trimester of gestation. The laminar blood flow from the maternal spiral artery changes to a turbulent flow upon entrance into the intervillous space. **Figure 1.** Potential route of maternal blood flow into the intervillous space during first trimester. During early placental development, maternal blood flow in maternal uterine spiral arteries is obstructed by extravillous trophoblast plugs. However, parts of maternal blood (red arrows) can pass through narrow intertrophoblastic gaps when trophoblast plugs begin to dissolve during first trimester of gestation. The laminar blood flow from the maternal spiral artery changes to a turbulent flow upon entrance into the intervillous space.

#### During human reproduction, even the preimplantation embryo, including the morula and later on the early blastocyst, is considered to be subject to fluidic flow forces generated by peristalsis of the **2. Flow Culture Approaches in Trophoblast Research**

fallopian tube. However, at this early phase of conception, the zona pellucida — a thick glycoprotein layer, which surrounds the oocyte to allow only species-specific fertilization and persists up to the early blastocyst stage — is suggested to protect the early embryo from harmful mechanical forces [9]. Only with the onset of intervillous perfusion, hemodynamic forces affect the phenotype and physiology of extravillous trophoblasts in invaded uteroplacental spiral arteries as well as villous trophoblasts covering placental villi. Failure of adaptions to this fluid shear stress is suggested to contribute to pregnancy disorders, including fetal growth restriction, which manifests upon impaired spiral artery remodeling, high vascular resistance, and placental hypoperfusion [7]. **2. Flow Culture Approaches in Trophoblast Research**  While in the early twentieth century basic principles for plant and animal cell cultures in vitro were developed (see comprehensive overview by Magdalena Jedrzejczak-Silicka http://dx.doi.org/10.5772/66905), first reports about continuous-flow culture of mammalian cells date back to the 1950s, which describe cells grown in suspension [10]. Later on, in the late 1970s, flow culture of periodontium explants from adult mouse, even under different pO2 ranges, has been reported [11]. During the past twenty years, a remarkable body of work has been devoted to adherent cell culture in microfluidic channels, which usually are designed in micron length-scales and are developed to generate well-defined microenvironments with various patterns of fluidic flow (pulsatile, steady, or oscillatory) [12]. By using such fluidic cell culture systems, a wide panel of different cell types, including stem cells, fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, hepatocytes, cancer cells, and neuronal cells have been analyzed [13]. In addition, human- and While in the early twentieth century basic principles for plant and animal cell cultures in vitro were developed (see comprehensive overview by Magdalena Jedrzejczak-Silicka http://dx.doi.org/ 10.5772/66905), first reports about continuous-flow culture of mammalian cells date back to the 1950s, which describe cells grown in suspension [10]. Later on, in the late 1970s, flow culture of periodontium explants from adult mouse, even under different pO2 ranges, has been reported [11]. During the past twenty years, a remarkable body of work has been devoted to adherent cell culture in microfluidic channels, which usually are designed in micron length-scales and are developed to generate well-defined microenvironments with various patterns of fluidic flow (pulsatile, steady, or oscillatory) [12]. By using such fluidic cell culture systems, a wide panel of different cell types, including stem cells, fibroblasts, endothelial cells, osteoblasts, smooth muscle cells, hepatocytes, cancer cells, and neuronal cells have been analyzed [13]. In addition, human- and animal-derived trophoblasts were subjected to fluidic flow culture, using rather diverse experimental setups (Tables 1 and 2). While used trophoblast-derived cell lines include the choriocarcinoma cell lines JAR [14], JEG-3 [15], and BeWo [16,17], as well as the Simian Virus 40 (SV40)-transformed trophoblast cell lines HTR-8/SVneo and SGHPL-4 [14,18], primary trophoblasts have been used after isolation from first trimester [19,20] and term placenta [21–23]. Besides human trophoblasts, primary macaque trophoblasts [23,24] have been used due to anatomical similarities between human and macaque placental tissues. Moreover, rabbit trophoblast progenitors derived from blastocyst obtained four days post coitum from New Zealand White female rabbits extend the long list of trophoblast cells used for fluidic flow experiments [17].

animal-derived trophoblasts were subjected to fluidic flow culture, using rather diverse experimental setups (Tables 1 and 2). While used trophoblast-derived cell lines include the choriocarcinoma cell lines JAR [14], JEG-3 [15], and BeWo [16,17], as well as the Simian Virus 40 (SV40)-transformed trophoblast cell lines HTR-8/SVneo and SGHPL-4 [14,18], primary trophoblasts have been used after


**Table 1.** Overview of flow culture approaches to study trophoblast motility.

human umbilical vein endothelial cells (HUVECs), gestational day (GD), uterine microvascular endothelial cells (UtMVECs), extravillous trophoblasts (EVTs). \* circulating flow loop; \*\* one-time inlet-to-outlet flow system;

According to literature, trophoblasts have been subjected to a rather broad range of different flow rates and shear stress values, ranging from the µl- to ml/min scale and 0.001-30 dyn/cm<sup>2</sup> , respectively. These variations in the experimental setup may reflect the general heterogeneity in blood flow through different vessel segments, with a mean shear stress of approximately 7.5 dyn/cm<sup>2</sup> in large veins, about 15 dyn/cm<sup>2</sup> in large arteries, and 30 dyn/cm<sup>2</sup> within venules and arterioles [22]. For the uteroplacental circulation, maternal blood flow through invaded uterine spiral arteries has been suggested to be 1–10 dyn/cm<sup>2</sup> , whereas the flow rate is drastically reduced to 0.001– 0.1 dyn/cm<sup>2</sup> after entrance into the wide cavity of the intervillous space, and is assumed to peak at roughly 2 dyn/cm<sup>2</sup> in some areas [16,25,26]. The shear stress exerted on different areas of the syncytiotrophoblast surface may vary, as the intervillous space is a highly asymmetric open space and chorionic villi show a very complex structure [25]. Therefore, different regions within the intervillous space (proximal or distal to the spiral arterial opening), and even different parts of a villous tree (free-floating or anchoring villi) may be faced with different dimensions of shear stress. Since fluid shear stress depends on the dimension and architecture of the vessel, design of the used cell culture dish is a critical aspect for the experimental setup. Depending on the addressed research questions, authors used various different systems ranging from commercially available 2D flow chambers to borosilicate glass capillary tubes and customized 3D micro-scale plastic ware solutions. Beside commercially and customized culture devices, the use of dextran microcarrier beads in combination with fluid shear stress produced by a rotating wall vessel bioreactor has been described to achieve a 3D flow culture model [27]. Moreover, different flow protocols, including open systems and closed circuits, different protein surface coatings (e.g., collagen type I [16,17,20,22–24], and fibronectin [14,18,28]) and different incubation times from only minutes [16] up to 96 h [17] and even 21 days [27] have been described.


**Table 2.** Overview of flow culture approaches to study trophoblast differentiation and fusion.

human villous trophoblasts (HVTs), rabbit trophoblastic stem cells (rTSCs), human umbilical vein endothelial cells (HUVECs), human brain microvascular endothelial cells (HBMECs), human primary placental villous endothelial cells (HPVECs), \* circulating flow loop; \*\* one-time inlet-to-outlet flow system; \*\*\* rotating wall vessel (RWV) bioreactor.

Thus, study questions, such as to how fluidic flow and shear stress influences different aspects of trophoblast physiology (e.g., differentiation and fusion (Table 2) and migration (Table 1)), should be addressed with a most appropriate setting of the flow system.
