Purification of HLA-G<sup>+</sup> Extravillous Trophoblasts from Human Term Placental Tissues for Phenotyping and Functional Analysis

The human maternal-fetal interface represents a unique immunological environment where maternal immune cells and fetal extravilllous trophoblasts (EVT) engage in direct cellular contact to establish and maintain immune tolerance. EVT exhibit a distinct immunophenotype characterized by the expression of tolerogenic molecules, including HLA-G, PD-L1, and PD-L2, which play pivotal roles in modulating maternal immune responses. In addition, human EVT express HLA-C that can be directly recognized by maternal NK and T cells and promotes their activation. Importantly, no orthologues of HLA-C or HLA-G are present in animal models used for research, and murine trophoblast lack MHC expression. Thus, investigating the phenotypic and functional properties of human EVT from placental tissues, under both physiological and pathological conditions, is essential for elucidating the mechanisms underlying pregnancy maintenance and pathophysiology of placental inflammation associated with preeclampsia, preterm birth, fetal growth restriction, and placenta accreta. This protocol outlines a comprehensive methodology for the isolation and characterization of primary human EVT from healthy term placental tissue as well as after preterm birth and preeclampsia. It includes: (1) dissection of placental and chorionic membrane tissues; (2) enzymatic digestion and the preparation of single cell suspensions; (3) EVT purification by FACS sort; (4) Phenotypic characterization by high-dimensional flow cytometry; and (5) short-term in vitro culture and co-culture with maternal immune cells for up to 96 h. Emphasis is placed on obtaining highly pure and viable EVT suitable for downstream applications such as immunological functional assays, protein and gene expression analysis. The implementation of this protocol enables a robust and reproducible platform for advancing understanding of EVT and maternal immune cell interactions and its implications in both healthy and complicated pregnancies.