The placenta is a temporary fetal organ essential for the survival of the fetus. It comprises trophoblasts, including extravillous trophoblasts (EVTs), which play a crucial role in adapting the human body to pregnancy. The absence of a stable, non-transformed proliferative EVT model is a gap that the group is working to fill. To address this, they generated different EVT cell lines from 3D cultures of trophoblastic cells isolated from chorionic villi of first-trimester human placentas. This study focuses on the characterization and comparison of two cell lines. qPCR analyses were conducted to quantify the expression of trophoblastic markers; immunofluorescence was used to assess the expression and localization of EVT-specific proteins, and flow cytometry was used to identify immunomodulatory antigens expressed on the cell surface. This work enabled the characterization of the two trophoblastic cell lines, laying the groundwork for developing a model to study EVTs more comprehensively.