Placental Barrier-on-a-Chip: Modeling Placental Inflammatory

Placental inflammation, as a recognized cause of preterm birth and neonatal mortality, displays extensive placental involvement or damage with the pre...
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Tissue Engineering and Regenerative Medicine

Placental barrier-on-a-chip: modeling placental inflammatory responses to bacterial infection Jianhua Qin, Yujuan Zhu, Fangchao Yin, Hui Wang, Li Wang, and Jingli Yuan ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.8b00653 • Publication Date (Web): 16 Jul 2018 Downloaded from http://pubs.acs.org on July 18, 2018

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Placental barrier-on-a-chip: modeling placental inflammatory responses to bacterial infection Yujuan Zhua,b,c, Fangchao Yinb,c, Hui Wangb,c, Li Wangb, Jingli Yuana, and Jianhua Qinb,c,d* a

College of Chemistry, Dalian University of Technology, Dalian 116024, China,

b

Division of Biotechnology, Key Laboratory of Separation Sciences for Analytical Chemistry, Dalian Institute of

Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China c

University of Chinese Academy of Sciences, Beijing 100049, China

d

CAS Center for Excellence in Brain Science and Intelligence Technology, Chinese Academy of Sciences,

Shanghai 200031, China *Correspondence should be addressed to J.Q. ([email protected]).

Abstract

Placental inflammation, as a recognized cause of preterm birth and neonatal mortality, displays extensive placental involvement or damage with the presence of organisms. The inflammatory processes are complicated and tightly associated with increased inflammatory cytokine levels and innate immune activation. However, the deep study of the underlying mechanisms was limited by conventional cell and animal models due to great variations in the architecture and function of placenta. Here, we established a microengineered model of human placental barrier on the chip and investigated the associated inflammatory responses to bacterial infection. The multilayered design of the microdevice mimicked the microscopic structure in the fetal-maternal interfaces of human placenta, and the flow resembled the dynamic environment in the mother’s body. Escherichia coli (E. coli), one of the predominant organisms found in fetal organs, were applied to the maternal side, modeling acute placental inflammation. The data demonstrated the complex responses including the increased secretion of inflammatory cytokines by trophoblasts and the adhesion of maternal macrophages following bacterial infection. Particularly, transplacental communication was observed between two

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placental cells, and implied the potential role of trophoblast in fetal inflammatory response syndrome in clinic. These complex responses are of potential significance to placental dysfunctions, even abnormal fetal development and preterm birth. Collectively, placental barrier-on-a-chip microdevice presents a simple platform to explore the complicated inflammatory responses in human placenta, and might help our understanding of the mechanisms underlying reproductive diseases. Key words: Placental barrier-on-a-chip, placental inflammation, bacterial infection, organ-on-a-chip

Introduction

Preterm birth, defined as birth before 37 week’s gestation, is a predominant cause of neonatal morbidity and mortality accounting for an estimated 1 million infant death every year1-2. These preterm newborns are at increased risk of fetal inflammatory response syndrome and sepsis, and face a series of short- and long-term severe complications during development3-6. In clinic, the preterm deliveries often occur in the case of placental inflammation, and these inflammatory processes are always accompanied with the placental damage with loss of function, release of inflammatory mediators inducing damage to fetal organs, and transplacental infection of the growing fetus7. Although these responses were complicated, the course of placental inflammation is commonly asymptomatic in the mother8. Furthermore, conventional cell monolayers and animal models are limited by great variations in the architecture and function of tissue, and the use of human placenta is inconvenient due to ethical concerns of tissue availability, and practical concerns of tissue storage and manipulation. Therefore, it is highly desirable to develop novel models to explore placental pathology in humans. Recently, the advances in bioengineering and microfabrication provide new chances for the development of in vitro disease models. In fact, human diseases are hugely complex due to a multitude of inter-related genetic variations and a variety of environmental factors, thus even completely different biological responses could be obtained in the same organ. For these reasons, it is highly challenging to

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develop in vitro models resembling every facet of human disease. Therefore, researchers take a synthetic approach to create complex in vitro experimental systems that recapitulate many characteristics that are critical for disease etiology and progression. At present, a series of organs-on-chips have been established, which recapitulated more and more features, such as the specialized tissue interface, dynamic flow, in the brain, lung, liver, gut, kidney, and many other human organs and tissues9-17, Specifically, a microengineered placental model has been established in recent studies15-16, which resembled key features of human placental barrier. By combining different cells and tissues, these novel in vitro models created the in vivo-like microenvironment to mimic human-specific pathophysiology, and enabled the analysis at molecular and cellular scale and the identification of new therapeutic targets within an organ-level context. Here, we adapted this approach to engineer a human placental barrier-on-a-chip microdevice resembling the near-physiological placental barrier, and further explored the placental inflammatory responses with bacterial infection. The established microsystem recapitulated cell types, multi-layered structure of placental barrier, and dynamic microenvironment in vitro. Such an engineered functional unit of human placenta was utilized as an experimental model to investigate inflammatory responses of placental tissue with exposure to E. coli, which is one of the most common bacteria associated with preterm birth.

Experimental

Cell culture

BeWo cells (human trophoblast cell line) was obtained from Cell Resource Center of Institute of Basic Medical Sciences, Chinese Academy of Medical Science, and maintained in DMEM/F12 medium (GE Healthcare) containing 20% fetal bovine serum (FBS, Gibco), 1% L-glutamine (Gibco), and 1%

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penicillin/streptomycin (Gibco). Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical vein18. HUVECs were cultured on the Collagen I-coated plate and maintained in ECM medium containing 5% FBS (Lonza). THP-1 cells were cultured in suspension in RPMI 1640 containing 10% fetal bovine serum (FBS), 1% L-glutamine, and 1% penicillin/streptomycin.

Microdevice fabrication

The multi-layered microdevice was fabricated using standard soft lithography techniques as described in our previous studies13-14. Briefly, poly(dimethylsiloxane) (PDMS) (Sylgard, Dow Corning) monomer was mixed with the curing agent (Dow Corning) at a weight ratio of 10:1, and the mixture was then cast on the molds made of SU-8 (MicroChem Corp.) for the preparation of the upper and lower layers with microchannels (width, 1.5 mm; length, 1.5 cm; height, 400 μm). The transparent semipermeable membrane with 0.4 µm pores (GE Healthcare) adhered to the bottom of the upper layer through the electrostatic interaction. Then PDMS as the glue, which was the mixture of PDMS base and curing agent at a weight ratio of 50:1, was evenly smeared on the membrane. After the curing of PDMS glue, the upper and bottom layers were bonded together following the plasma treatment. Finally, the multi-layer chip with the polycarbonate membrane was cured at 80 °C for 30 min.

Microfluidic cell culture

The assembled microdevice was firstly sterilized using UV irradiation. Following sterilization, microchannels were coated with Collagen I (0.1 mg ml−1 in ddH2O) contributing to cell adherence and growth. On day 0, HUVEC was harvested and resuspended in ECM medium at a density of ~2 × 106 cells per ml. These cells were then introduced into the lower microchannels, and microdevice was immediately inverted for the adherence of endothelial cells onto the lower side of the membrane. Specifically, the inlets and outlets of the lower microchannels were blocked using PDMS bar, preventing

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the outflow of cell suspension. After the incubation at 37 °C for 1 hr, BeWo cells, which were suspended in DMEM/F12 medium at a density of 4 × 106 cells per ml, were seeded onto the upper microchannels. Following the culture at 37 °C for 2 hr, unattached cells were washed away and the medium in the microsystem was changed to the mixture of ECM medium and DMEM/F12 medium (1:1). On day 2, DMEM/F12 and ECM medium was introduced into the upper and lower microchannels, respectively, using syringe pumps with the flow of 10 µl/hr. Differentiated THP-1 cells suspended in RPMI 1640 medium at a density of 1 × 105 cells per ml were perfused into the upper microchannel for 30 min at 40 µl/hr, and then was prewashed with PBS for several times before imaging. Differentiation of monocytes into macrophages Differentiation of THP-1 monocytes into macrophages was performed as described in the previous study19. Briefly, THP-1 cells were treated with 200 nM PMA for 3 days at first, and then cultured for another 5 days in fresh RPMI 1640 medium before use. Live cell staining CellTracker Green or Red (Life technologies) was used to stain live cells. HUVEC and BeWo cells were incubated with dye in basal medium without serum at 37℃ for 30 min. Cells were then washed with PBS for several times before imaging.

Bacterial transformation and infection

E. coli constitutively expressing green fluorescence protein (GFP) were cultivated in LB broth at 37 °C, 5% CO2 conditioned incubator with shaking for 12 hr. Arabinose (0.5 mg/ml) was introduced into the culture broth to induce the expression of the pGLO plasmid as demonstrated by the presence of GFP fluorescence. Bacteria cells were resuspended in DMEM/F-12 medium (density, ∼1.0 × 107 CFU/mL) without penicillin/streptomycin, and inoculated with trophoblastic epithelium in the upper microchannel. Placental cells were prepared for subsequent biological analysis after the inoculation with bacteria for 6

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hr under the static condition.

Real-time PCR

Total mRNA extraction and real-time PCR was performed as described in previous study14. Primer pairs used were listed in Table S1.

Immunohistochemistry

The details about immunohistochemical staining were described in our previous studies14. Primary antibodies used here were listed as follows: OCCLUDIN (mouse, Life technologies, 331594, 1:100), VE-CADHERIN (rabbit, CST, 2500S, 1:100), GLUT1 (rabbit, Proteintech, 21829-1-AP, 1:100). All staining images were gained using a confocal microscope (Olympus). Staining for microvilli Following culture for 72 hr, trophoblast cells grown in microfluidic devices were fixed with 4% paraformaldehyde for 10 min, and permeabilized with 0.25% Triton X-100 for 5 min after washing with PBS. Then samples were stained with Alexa 488-conjugated phalloidin (Biotium) for 30 min at room temperature as described by the manufacture after washing with PBS. The cell nuclei were counterstained with DAPI. Before inspection, samples were washed with PBS and mounted. The microvilli were viewed by a confocal microscope (Olympus). Live/dead cell assay The assay of live and dead cells in our microsystems was performed using the Live/Dead viability assay kit, Gibcao as described in product information by the manufacturer. Results and discussion

Reconstitution of placental barrier-on-a-chip

In vivo, placental barrier is a semipermeable multi-layered structure consisting of the trophoblastic

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epithelium in the maternal interface and the endothelium in the fetal interface, thereby separating the maternal and fetal circulation in placenta (Fig. 1a, b). Herein, we designed and fabricated a three-layer microdevice to create the placental barrier-on-a-chip, characterized by the co-culture of human trophoblasts (BeWo on the top) and human endothelial cells (HUVECs on the bottom) on opposite sides of a thin semipermeable membrane (Fig. 1c, d). Both trophoblasts and endothelial cells were cultured with perfusion system, resembling the dynamic flow environment in maternal and fetal blood circulation in human body. Specifically, the multi-layered design of this microsystem contributes to the co-culture of BeWo cells and HUVECs, resembling the multilayered physical structure in human placenta. A porous membrane sandwiched between two cell layers enabled the two key placental cells to grow and organize into a multilayered tissue, which recapitulate the native microarchitecture of the maternal-fetal interface. The design of this device makes it possible to culture different cells in compartmentalized chambers, as well as create the soluble microenvironment, facilitating the investigation of inflammatory responses in maternal or fetal part exclusively. Following introduction into the microchannels, the epithelial and endothelial cells were attached to the ECM-coated membrane and grew with perfusion system (Fig. 2a). These placental cells covered the entire surface of the porous membrane and finally formed fully confluent monolayers in both maternal and fetal parts. To further verify the integrity of the barrier, trophoblast cells were stained for OCCLUDIN, which played an essential role in tight junction stability and barrier function20, and endothelial cells were stained for VE-CADHERIN, which was known to be particularly important for maintaining the integrity of intercellular junctions in the endothelial barrier21. As shown in Fig. 2b and 2c, the trophoblastic epithelium and endothelium were positive for OCCLUDIN and VE-CADHERIN respectively, which localized clearly at the cell–cell junctions between cells. Such confluent tight monolayers were required

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for subsequent experiments to study various responses of placental barrier. Formation of polarized placental barrier Human placental cells constantly experience shear forces from the transportation of oxygen, nutrients, and waste products between maternal and fetal blood. By adapting to the microenvironment, trophoblastic cells in placenta develop microvilli to maintain cell and tissue functions. Microvilli, as another vital structural feature of placental barrier, is actually the membrane protrusions present on the apical surface of trophoblasts22. These microvilli could sense and interact with surrounding fluid microenvironment in vivo23, and enable polarized localization of various functional membrane proteins, thereby critical for the extensive cellular and tissue functions24. Using this microdevice, the trophoblasts in the upper microchannel formed the characteristic microvilli with the presence of fluid flow (Fig. 2d and Fig. S1), suggesting the essential role of dynamic flow in the cell morphology of trophoblasts. Confocal microscopy of endothelial cells stained for F-actin also revealed the elongated cellular morphology under the flow at high resolution (Fig. 2e). These observations implied the near-physiological features of our placental barrier model. Glucose transfer across the human placenta (maternal to fetal) is crucial to sustain the fetal growth, and this process is mediated by the GLUT1 glucose transporters, which is found in the microvillous trophoblastic layer25 and asymmetrically distributed (microvillous>basal)26-27. Here, immunofluorescence staining was performed to verify the pattern of GLUT1 expression in this microsystem. As shown in Fig. 2f, GLUT1 transporters were highly expressed in the apical microvilli (maternal side) compared to the basal part (fetal side) in trophoblasts, and such the varied expression might facilitate the maternal-to-fetal transfer of glucose during gestation. Considering the importance of microvilli in the regulation of placental function, these observations illustrate that our microsystem might be available to construct placental disease models in vitro based on

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the reproducible structural and physiological phenotypes. Inflammatory responses of placental barrier to bacterial infection As bacterial infections are associated with the acute inflammation of the placental membranes, we next explored whether the placental barrier-on-a-chip could be utilized to model these inflammatory responses in vitro. Key hallmarks of placental inflammatory diseases are associated with the compromise of the placental barrier, which are believed to result from complex pathological interplay among the placental epithelium, microorganisms, and maternal immune cells. To investigate how microorganisms lead to placental inflammation, we cocultured Gram-negative E. coli, a nonpathogenic strain labeled with fluorescent protein GFP (GFP-EC), with the placental barrier on the chip (Fig. 3a). As observed in fluorescent images in Fig. 3b, bacteria proliferated rapidly after the inoculation in the microsystem for 6 hr. Studies have demonstrated that E. coli could release lipoglycans, peptidoglycans, and cell membrane products that could induce the acute inflammation of the placental membranes28. As such, inflammatory cytokines were examined at mRNA level in trophoblast cells (Fig. 3c). Following the inoculation with E. coli, trophblasts spontaneously produced a much higher amount of inflammatory cytokines including Interleukin-1α (IL-1α), IL-1β, and IL-8. As a predictor for the identification of asymptomatic intrauterine infection in clinic29, IL-6 was also significantly upregulated in our micromodel of placental inflammation. Similar changes were also observed in IL-8 expression, which is a chemotactic and activating factor for the recruitment of inflammatory cells and play important roles in host defence mechanisms30. In vivo, IL-1α plays one of the essential roles in the regulation of the immune responses, and activates tumor necrosis factor-α (TNFα), which was consistent with the upregulated expression of both IL-1α and TNFα in this model. Clearly, the underlying epithelium was activated with the presence of these microorganisms.

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Trophoblast apoptosis is a vital mechanism of defense against bacterial infection, which might trigger membrane rupture31. Thus, placental cell death was assessed using live/dead cell assay after exposure to GFP-EC for 6 hr. As indicated in Fig. 3d and 3e, the GFP-EC bacteria triggered significant cell death in both trophoblasts and endothelial cells, mimicking cell death during membrane rupture observed in both other in vitro studies and animal models. These data mount the characteristic inflammatory lesions to E. coli infection in the placenta, and might be highly correlated with placental dysfunction resulting in adverse obstetrical outcomes. Activation of maternal macrophages to bacterial infection According to previous reports, the cross-talk between trophoblast and maternal macrophages is essential during the course of gestation,32-33 and dysfunctions in this communication system are tightly related to the pregnancy pitfalls34. Therefore, the inflammatory responses of macrophages towards bacterial infection were further examined based on this microdevice (Fig. 4a). As illustrated above, trophoblasts showed the increased inflammatory molecule levels, including IL-1α, IL-1β, IL-6, IL-8 and TNF-α. These chemokines might promote the arrival of immune cells in the maternal circulatory system to the placenta35, thereby the adherence of macrophages to the trophoblast layer was tested here (Fig. 4b). Following the inoculation with E. coli, human macrophages (THP-1) were introduced onto the trophoblast layer on the placental barrier-on-a-chip, and more macrophages were observed to be attached to the epithelium (Fig. 4c), suggestive of the activated maternal innate immune system in the bacteria-infected placenta. The stimulation of maternal macrophages might result from the interplay of risk factors including inflammatory mediators and cell apoptosis. Secretion of inflammatory factors by fetal endothelium to bacterial infection Placental inflammation is frequently accompanied with the fetal systemic inflammatory responses characterized by elevated levels of inflammatory cytokines in the fetal circulation36. Herein, we focused

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to explore the inflammatory responses of fetal vessels with bacteria in the maternal side. coli is a rod-shaped bacterium with approximately 0.5 μm in width and 2 μm in length37, which could not cross the porous membrane (pore size, 0.4 μm) in the microdevice. The co-culture microsystem with such a porous membrane allows paracrine signaling, a form of cell-to-cell communication altering the behavior of nearby cells. To investigate the transplacental communication between trophoblasts and endothelial cells, the inflammatory lesions in fetal vessels were thereby examined based on the expression of related inflammatory cytokines in the presence or absence of trophoblasts. As shown in Fig. 5a, products released by E. coli could stimulate the inflammatory responses in the endothelium, but with the presence of trophoblasts, the expression of inflammatory cytokines were further increased. This result suggested a significant association between trophoblastic cells and the release of cytokines from fetal endothelial cells, thereby the influence of bacterial infection on the integrity of endothelial monolayer was next examined in the presence or absence of trophoblasts. VE-cadherin was stained in fetal endothelial cells, and displayed the disrupted endothelial barrier, particularly in the presence of trophoblasts (Fig. 5b). Together, these results suggested the transplacental communication between two types of placental cells with bacterial infection, and the potential role of trophoblasts in fetal inflammatory response syndrome, which might be much closer to placental inflammation of bacterial origin in the body.

Conclusions

Herein, we have constructed a dynamic placental barrier-on-a-chip microdevice, which allowed the investigation of complicated inflammatory responses in human placenta with bacterial infection. The multilayered design of the microdevice allows the co-culture of trophoblasts and endothelial cells on the opposite sides of the porous membrane, resembling the functional unit of human placenta. By harnessing the natural fluid, shear environment was generated in both maternal and fetal sides. The exposure to E. coli in trophoblasts recapitulated the acute bacterial infection in the setting of the chorion.

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The intermediate porous membrane with 0.4 μm pore size contributes to keep the bacteria in the side of trophoblastic cells, thereby allowing to examine the inflammatory responses of endothelial cells with bacterial infection in the maternal side. According to our analysis, bacterial infection stimulated the secretion of inflammatory cytokines in maternal side and induced the activation of maternal macrophages. At the maternal-fetal interface, the maternal blood is in direct contact with the placenta, and the production of inflammatory factors from trophoblasts activate circulating macrophages, which are principle defense against the bacteria and central to the innate immune responses38. Furthermore, even without the transmission of microorganisms to the fetus, endothelial cells in fetal vessels were stimulated to secret inflammatory cytokines, and the response was exaggerated when co-cultured with trophoblasts on the chip, which demonstrated the transplacental communication between two key placental cells. Collectively, our placental inflammation model on the chip resembled the complicated observations in clinic, including the release of inflammatory mediators, and the adhesion of maternal monocytes. In particular, the transplacental communication were investigated, and implied the potential role of trophoblasts in fetal inflammatory response syndrome in clinic. All these responses are often associated with placental dysfunctions and even abnormal fetal development, and demonstrated that this microengineered model might offer a powerful platform for in vitro investigation of bacterial infection in human placenta. Despite the great potential of our model, there is still room for considerable improvement. In recent studies, placental cells including trophoblasts and endothelial cells could be efficiently differentiated from pluripotent stem cells38-39, which hold great promise in the field of developmental biology. The incorporation of these cells could advance the development of placental model mimicking various pathological conditions during gestation. Additionally, to establish a near-physiological model, one approach is to incorporate additional factors such as more physiologically relevant cells. In vivo, due to

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the secretion of inflammatory factors from the placenta, monocytes are usually activated together with other inflammatory cells including granulocytes and T cells. The cross-talk between bacteria and varied immune cells contributes to construct more predictive models, which could accurately reflect how our own body responds to pathogens. Further insight into the role of these cells under this condition may also lead to a better understanding of the complicated inflammatory responses including the systemic and local changes in placenta. We envision that more complex and realistic placental model could be built to accurately reflect responses in vitro through the incorporation of various microenvironmental factors, such as physiochemical factors, mechanical cues, and physiologically relevant cells, thereby greatly contributing to our understanding of human reproductive health and disease.

Supporting Information

Primer pairs used to examine mRNA expression of inflammatory factors. F-actin staining in BeWo cells under the static condition.

Acknowledgements

This research was supported by the Strategic Priority Research Program of the Chinese Academy of Sciences (No. XDA16020900, XDPB0305), Key Program of the Chinese Academy of Sciences (KFZD-SW-213), National Key R&D Program of China (No. 2017YFB0405400), National Nature Science Foundation of China (No. 91543121, 81573394, 31671038), Innovation Program of Science and Research from the DICP, CAS (DICP TMSR201601). We thank Professor Jinyi Wang (Northwest A&F University, China) for kindly providing fluorescence labeled E. coli.

Conflict of interest

There are no conflicts of interest to declare.

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Graphic for manuscript

Fig. 1 Design and assembly of the multilayered chip mimicking human placental barrier in vitro. a, The placenta is an organ that connects the developing fetus to maternal uterine via the umbilical cord. Bacteria commonly pass from the vagina or cervix into the uterus, and vulnerably cause the inflammation of the placental membrane. b, A cross-sectional view of the placenta in human body. The maternal blood comes into direct contact with the fetal chorion, allowing an exchange of gases and nutrients to take place. The chorionic villus absorb nutritive materials from uterine for the growth of the embryo. c, The schematic of the human placental barrier, which acts as the interface between the mother and fetus. The barrier is composed of the outer trophoblast layer and the inner fetal endothelial cell layer. Such the barrier plays an essential role in the maternal and fetal circulations, and governs the cross talk between the maternal and fetal microenvironments. d-e, The diagram of placenta-on-a-chip microdevice including the top layer (maternal channel), the middle porous membrane and the bottom layer (fetal channel). The microdevice was made of biocompatible PDMS. f, Placental barrier was

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constructed on the chip, with BeWo epithelial layer and HUVEC layer on the opposite sides of the membrane. The flow resembled the dynamic environment in placenta in vivo.

Fig. 2 The characterization of placental barrier-on-a-chip in vitro. a, Trophoblasts and endothelial cells formed confluent cell layers on the opposite sides of the porous membrane (pore size, 0.4 µm). b, Immunohistochemical staining for Occludin in BeWo cells, which is important in tight junction stability and barrier function. DAPI marks nuclei (blue). c, Immunohistochemical staining for VE-cadherin in HUVEC, which is required for maintaining the integrity of the intercellular junctions in the endothelial barrier. d, F-actin staining (green) for the widespread microvilli in BeWo trophoblastic cells. e, Staining for for F-actin with Alexa-Fluor-488–phalloidin (green) in HUVEC. White arrows indicate the flow. f, Staining for GLUT1 transporters in trophoblast cells (upper) and the cross-sectional view showing the asymmetric localization of GLUT1 transporters to the apical surface. Scale bars, 100 µm (a), 30 µm (b), 50 µm (c-e), 10 µm (f).

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Fig. 3 The bacterial infection in the placental barrier on the chip. a, The schematic of bacterial infection in placenta. b, E. coli proliferated rapidly 6 hr after being inoculated in the channel of trophoblast cells. Representative images of GFP-expressing bacteria (green) in the microsystem at 0 and 6 hr. c, Quantitative real-time PCR analysis of the expression of inflammatory genes in BeWo cells stimulated with E. coli. P values were calculated based on 3 experiments using unpaired, two-sided Student’s t-test. *, P < 0.05, ***, P < 0.001. d, Examination of cell viability after infection with E. coli. Dead cells (red), live cell (green). e, Quantification of dead cells in BeWo trophoblastic cell layer and endothelial cell layer. The data was calculated from 4-5 images. n=3 independent experiments. *, P < 0.05, ***, P < 0.001. Scale bars, 100 µm (b, c), 50 µm (e).

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Fig. 4 THP-1 monocyte adhesion to trophoblast cells with exposure to E. coli. a, Schematic representation of the maternal macrophages in the placenta at the maternal-fetal interface during bacterial infection. b, BeWo trophoblastic cells were inoculated with E. coli for 6 hr following washing with PBS. BeWo cells were then co-cultured with THP-1 (red) for 30 min. Microphotographs (from 3 independent experiments) were obtained using a fluorescence microscopy. c, The number of adherent THP-1 cells was calculated in 4-5 random images, and the statistical test was calculated based on 3 experiments using unpaired, two-sided Student’s t-test. ***, P < 0.001. Scale bars, 100 µm.

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Fig. 5 Inflammatory responses of fetal endothelial cells on the chip. a, Quantitative real-time PCR analysis of the expression of inflammatory genes in endothelial cells. The microsystem with endothelial cells in the absence of bacteria was served as a control group. For another two groups, bacteria were introduced into the maternal channel in the absence (EC monoculture + E. coli) or presence (Coculture + E. coli) of BeWo, and HUVECs in fetal channels were harvested for analysis. P values were calculated based on 3 experiments using unpaired, two-sided Student’s t-test. *, P < 0.05, **, P < 0.01, ***, P < 0.001. b, Immunohistochemical staining for VE-cadherin in endothelial cells in three groups. Scale bars, 50 µm.

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For Table of Contents Use Only Placental barrier-on-a-chip: modeling placental inflammatory responses to bacterial infection Yujuan Zhu, Fangchao Yin, Hui Wang, Li Wang, Jingli Yuan, and Jianhua Qin*

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