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Introduction: Preeclampsia and IUGR have been linked to improper trophoblast migration that impairs maternal spiral artery transformation minimizing vessel dilation and therefore restricting blood flow to the developing fetus. Endothelial cells are thought to play an essential role in directing trophoblast migration and transformation, although the mechanism by which this occurs is not fully understood. We have developed a novel in vitro system that evaluates endothelial-trophoblast interaction and signaling in a three-dimensional environment. Methods: An immortalized human endometrial endothelial cell line (HEEC) (Krikun et al., 2004) and first trimester trophoblast cell lines HTR-8 and Swan.71 were used. Endothelial transformation into vessel-like structures occurred in Matrigel Basement Membrane Matrix. Cell membrane linker dye and OpenLab Image Analysis software were used to monitor trophoblast migration and transformation. Cytokine and chemokine production was determined using a multiplex bead assay. Results: Upon differentiation in matrigel, endothelial cells undergo a shift in cytokine production characterized by an increased secretion of chemokines. First trimester trophoblast cells migrate toward the differentiated endothelium, and reach it within 4–8 hr. Complete replacement of the endothelium is seen by 48 hr. The transformed tubes maintain structure and remain functional for several weeks allowing for further co-culture studies. Conclusion: We report the characterization of a novel three-dimensional in vitro system of trophoblast–endothelium cell interaction. We found significant changes in the phenotype (cytokine expression and LPS responses) of endothelial cells upon differentiation in matrigel as compared to the monolayer culture. These changes may be necessary for the endothelium to direct trophoblast migration. Furthermore, this model represents an ideal co-culture system to evaluate the role of additional cell types, e.g. immune cells, involved in the process of spiral artery transformation.