Isolation of pure primary term human cytotrophoblasts and their differentiation into syncytiotrophoblast-like cells as an ex vivo model of the human placenta.

The placenta plays a fundamental role in fetal growth and maintenance of pregnancy. Its cellular components include a large multinucleated syncytiotrophoblast (STB) and its progenitor, cytotrophoblasts (CTBs), both of which perform vital functions in the human placenta. Primary cytotrophoblasts isolated from term human placentas that spontaneously fuse and differentiate into syncytiotrophoblast-like cells in vitro have been utilized to investigate the functions of the syncytiotrophoblast and placenta with multiple modifications. Although recent advances have enabled the use of trophoblast stem cell-derived organoids as a model for villous trophoblasts, primary CTBs offer several advantages, including spontaneous differentiation, easy access to materials (from term-delivered human placentas), and simple methodology. Here, we present a precise step-by-step process for isolating pure CTBs from term human placenta based on previously reported placenta digestion, density centrifugation, and CTB purification using anti-HLA-A, B, C antibody. Subsequently, we provide a method to improve CTB viability and differentiation into STB-like cells using epidermal growth factor (EGF) and a ROCK inhibitor (Y-27632) that ensures long-term and stable cultures without altering their proliferation. Because these cells can grow on standard tissue culture plates, this model can be easily utilized for various placental investigations, including innate immune responses, drug resistance, and STB metabolism. Employing this approach considerably enhances our understanding of placental functions, which are key to maternal and offspring health.