Isolation and Culture of Term Human Cytotrophoblast Cells and In Vitro Methods for Studying Human Cy

Human primary cytotrophoblast cell culture is a very useful model to study the endocrine and immunological functions of syncytiotrophoblasts, as well as the ion exchange between the mother and her fetus, like calcium. In this chapter, we expose the procedure to (1) isolate and purify the cytotrophoblast cells from human term placenta and (2) study syncytiotrophoblast calcium uptake. First, the methodology is based on the enzymatic dissociation of villous placental tissue, followed by Percoll gradient separation. Purity is assessed by flow cytometry using staining against cytokeratin-7, protein specific for trophoblast cells. Cell proliferation is evaluated by a Thiazolyl Blue Tetrazolium Bromide (MTT) assay, hormonal secretion is measured by enzyme-linked immunosorbent assay (ELISA), and fusion is estimated by immunofluorescence using staining against desmosomal proteins. Second, we describe the calcium uptake experiment using the cytotrophoblast cells in culture.