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High School Poster

HS Poster

Poster 85 - Human placental blood vessels-on-a-chip to investigate acute vascular toxicity of bi-sphenol A and bisphenol S during human pregnancy

Saturday, October 26, 2024
10:00 AM – 12:00 PM EST
Location: Exhibit Hall E, F & G

Introduction: Bisphenol A (BPA) is an industrial chemical widely used to manufacture a variety of plastic-based consumer products. BPA is known as an environmental material with negative health implications, but much remains to be learned about its adverse effects on human pregnancy. Evidence collected from human clinical specimens has demonstrated the ability of BPA in the maternal blood to cross the placental barrier and enter fetal circulation. It remains unknown, however, whether and how BPA affects tissues in the fetal compartment during the progression of pregnancy.
Here we describe interdisciplinary efforts to address this important knowledge gap with the specific goal of investigating how BPA affects the vasculature of the umbilical cord, which is an essential element of pregnancy that permits blood transport between the placenta and the fetus. Motivated by the difficulty of studying this and other similar questions through human subject research, we present an alternative approach based on in vitro culture of primary human cells in microfabricated devices to create a bioengineered model of human umbilical cord blood vessels. We demonstrate the proof-of-principle of i) how this system enables in vitro modeling of vascular exposure to BPA-containing blood flow in the human umbilical cord and ii) how this specialized model can be used to assess the potential of BPA to induce acute vascular toxicities. Furthermore, we examine the effects of bisphenol S (BPS) to provide preliminary in vitro human data that may be useful for further evaluation and validation of its capacity as a safer substitute for BPA.

Materials and

Methods: Our bioengineered model was created in a poly(dimethylsiloxane) (PDMS) microdevice fabricated using soft lithography. The device consists of a central culture chamber flanked by two microchannels (Figure 1A). To generate blood vessels, we injected a mixture of primary human umbilical vein endothelial cells (HUVECs), fibroblasts, fibrinogen, and thrombin into the central chamber to embed the cells in a fibrin hydrogel and grow them in the 3D environment with media supply from the side channels (Figure 1B). This culture configuration allowed us to mimic the spontaneous process of new blood vessel formation during tissue development or regeneration, known as vasculogenesis, in which endothelial cells self-assemble into a network of interconnected, perfusable blood vessels (Figure 1B).

After 5 days of culture, our model was perfused with culture media containing 1, 200, or 500 μM of BPA or BPS to simulate low-, intermediate-, or high-level exposure conditions, respectively. Acute vascular toxicity was assessed by i) measuring changes in vascular architecture, ii) imaging of caspase 3/7 activity to detect cellular apoptosis, iii) enzyme-linked immunosorbent assay (ELISA) analysis of device effluent to quantify cellular release of lactate dehydrogenase (LDH), and iv) ELISA of device effluent to measure the production of interluekin-8 (IL-8) selected as a representative pro-inflammatory cytokine. These endpoints were measured 1, 24, and 48 hours after the initiation of bisphenol exposure. Quantitative data obtained from these analyses were presented as mean ± SD with n = 3, and statistical analysis was performed using the Graph Pad Prism 10 software and two-way ANOVA test.

Results, Conclusions, and Discussions: Using our culture method, we were able to form a cell-laden fibrin scaffold in the central chamber (Figure 1C) in which HUVECs self-organized into a network of interconnected, perfusable tubular structures during 5-day culture (Figures 1D,1E). The tissue constructs perfused with 1 μM BPA showed no significant changes in overall vascular architecture, and the individual vessels appeared to maintain their structural integrity regardless of exposure duration (Figure 2). In contrast, the intermediate and high-level exposure conditions led to noticeable structural deterioration in a dose-dependent manner after 24 hours of exposure, as illustrated by a loss of vascular connectivity, reduced vascular density, and decreased vessel length (Figure 2). These changes became more prominent over time, resulting in disintegration of the vascular networks after 48 hours (Figure 2).

Supporting these observations, caspase and LDH assays yielded no changes at 1 μM when compared to untreated and vehicle controls, but both endpoints were significantly elevated when the model was exposed to 200 μM for 24 hours, which was further increased at 48 hours (Figure 3). The same trend was observed at 500 μM, with the difference that the extent of BPA-induced caspase signaling and LDH release was greater (Figure 3). Interestingly, our data revealed significantly higher levels of IL-8 even at 1 μM after 24 hours of exposure (Figure 3C), and the production of this cytokine continued to increase over time (Figure 3C). Taken altogether, these results indicate that i) 1 μM BPA does not cause acute vascular injury in our model, nor does it lead to cellular apoptosis, but the low-level exposure condition can induce acute vascular inflammation and that ii) the higher BPA concentrations can cause a rapid loss of vascular tissue integrity. Importantly, our study using BPS showed that depending on its dose, BPS can be less toxic than BPA in our model, but these chemicals may produce similarly deleterious effects in terms of their potential to induce vascular inflammation (Figures 4, 5).

We believe that our technology may provide a potentially powerful platform for preclinical studies of placental and fetal toxicities of bisphenols and other chemicals during human pregnancy.

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