A bioengineered model of human placental exposure to heavy metals during pregnancy

Introduction:

Introduction: Prenatal exposure to toxic heavy metals poses significant threats to fetal development and has been associated with various pregnancy complications. Studying the adverse effects of heavy metals on human pregnancy, however, is challenging due to the difficulty of human subject research and the limited capacity of animal models to accurately represent the human reproductive system. Here, we present an alternative approach that leverages the design flexibility, controllability, and scalability of bioengineered human reproductive tissues to enable experimental simulation and in-depth investigation of placental exposure to heavy metals in maternal circulation. Central to this method is an in vitro analog of the maternal-fetal interface and its dynamic tissue-specific environment constructed using primary human placental cells grown in a microengineered device. Using cadmium as a representative toxicant, we demonstrate the proof-of-concept of emulating the human placental barrier subjected to the flow of cadmium-containing maternal blood to show how this model can be used to capture and examine adverse biological responses and impaired tissue function in both the maternal and fetal compartments. Moreover, we present a mechanistic study of maternal-to-fetal cadmium transport in this system to reveal the protective role of efflux membrane transporters against cadmium-induced toxicity. Finally, we describe metabolomic analysis of our microphysiological system to demonstrate the feasibility of discovering metabolic biomarkers that may potentially be useful for detection and monitoring of placental dysfunction due to cadmium exposure.

Materials and

Methods:

Methods: We used soft lithography to fabricate a compartmentalized elastomeric device consisting of two overlapping layers of microfabricated chambers separated by a thin semipermeable membrane (Figs. 1a, 1b). Following fabrication and sterilization, the device was used to grow i) primary villous endothelial cells and placental fibroblasts (Amnion Foundation) isolated from human placentas in fibrin hydrogel in the lower layer representing the fetal compartment and ii) primary human trophoblast cells (Amnion Foundation) in the upper maternal chamber at physiological oxygen tension (5%). The microengineered human placental tissue construct was cultured and maintained under perfusion conditions using computerized syringe pumps. To simulate placental exposure to cadmium in this model, the syncytialized maternal tissue in the upper chamber was subjected to a flow of culture media containing cadmium at varying concentrations (0.5-10 µM). Responses of our model were analyzed using various techniques, including microfluorimetry, ELISA, RT-PCR, and metabolomics.

Results, Conclusions, and Discussions:

Results: Co-culture of placental fibroblasts and endothelial cells in the fetal chamber of our device for 7 days allowed the endothelial cells to self-assemble into a three-dimensional (3D) network of interconnected, perfusable endothelial tubes reminiscent of the fetal microvasculature in the vascularized stroma of chorionic villi (Figs. 1c, 1d). The primary trophoblasts cultured on the upper side of the membrane proliferated to form a confluent monolayer, after which they underwent spontaneous fusion to form a syncytialized tissue layer within 3 days (Fig. 1f) that produced increasing amounts of the pregnancy hormone, beta human chorionic gonadotropin (β-hCG) (Fig. 1h) and exhibited the functional capacity to regulate maternal-to-fetal glucose transfer at physiological rates (Figs. 1i, 1j). When this model was exposed to CdCl2, we observed concentration-dependent deleterious responses in both maternal and fetal tissues, as evidenced by reduced cell viability (Fig. 2a), increased secretion of proinflammatory cytokines in the effluent of maternal and fetal compartments (Fig. 2b), and compromised barrier function. Importantly, incorporation of Hofbauer cells, which are placental villous macrophages of fetal origin, into the vascularized stroma of our model accentuated the cadmium-induced inflammatory responses (Fig. 2c-d). Furthermore, metabolomic analysis revealed previously unknown pathways that can be significantly altered by low- and high-dose cadmium exposure (Fig. 2f-j).

Conclusions: The placenta-on-a-chip provides a potentially powerful in vitro platform for studying adverse biological effects of cadmium and other environmental toxicants on the essential organ of pregnancy.

Acknowledgements (Optional):

Funding: This was supported by funding provided by the NIH (R01ES029275, P30ES005022, UL1TR003017, P30 Grant - Center of Excellence in Environmental Toxicology (CEET) - P30ES013508.