Extracellular matrix properties, interstitial flow, and VEGF gradients shape trophoblast behavior in a pumpless Trophoblast Invasion-on-Chip (TIoC)

Placental trophoblast invasion into the maternal endometrium is a critical step in establishing the maternal-fetal interface during implantation. In vitro, this complex process can be partially modeled using a Trophoblast Invasion-on-Chip (TIoC) platform. In this study, we used an in-house-developed, pumpless, recirculating "loop-in-loop" microfluidic device that generates interstitial flow (IF) within a tissue compartment to optimize indirect co-culture of cytotrophoblast BeWo cells with human umbilical vein endothelial cells (HUVECs). Eight extracellular matrix (ECM) conditions were tested, including fibrin-based matrices and laminin/collagen-based composites. Pure fibrin ECMs were less porous and stiffer than laminin/collagen matrices; however, mixed ECMs exhibited increased softness and porosity due to structural heterogeneity. BeWo cells demonstrated enhanced migration in fibrin-based mixtures and upregulated markers associated with the extravillous trophoblast phenotype. In contrast, HUVECs did not exhibit migration under any ECM condition. Differences in trophoblast migration were associated with ECM physical properties, biochemical composition, and degradability, and vascular endothelial growth factor (VEGF) diffusion within the matrices. Additionally, the microfluidic device generated unidirectional interstitial flow and established a negative VEGF gradient, which may in part arise from BeWo cell secretion, together with ECM porosity, influenced BeWo cell migration and differentiation in an ECM-dependent manner. Complementary experiments using primary cytotrophoblasts (CTBs) revealed a more restricted, ECM-dependent migration pattern, with a preference for Fibrin over more complex matrices such as Fibrin-Geltrex. Overall, this study demonstrates a clear relationship between trophoblast behavior the, physical and biochemical ECM properties, and the microfluidic flow dynamics. These findings highlights the importance of tuning biochemical, mechanical, and fluidic cues to better elucidate the biomechanical drivers of trophoblast invasion.