Isolation, purification and in vitro differentiation of cytotrophoblast cells from XXXX term placenta.

@entity1 @entity5962 @entity1195 @entity2430 @entity2279 @entity11772 @entity5822 @entity2277 @entity6393
BACKGROUND: The syncytialization of cytotrophoblast cells to syncytiotrophoblast is central to @entity1 placental transport and hormone production. Many techniques for in vitro study of this process have been proposed and new investigators to the field may find the literature in the field daunting. Here, we present a straightforward and reliable method to establish this important model using modern but readily available tools and reagents. METHODS: Villous cytotrophoblast cells are obtained from term placenta using mild enzymatic degradation, Percoll gradient centrifugation, negative magnetic cell sorting using an antibody against classical major histocompatibility complex molecules and in vitro culture on a matrix-coated growth surface. RESULTS: The purity of isolated cytotrophoblast cells exceeds 98  % as assessed by @entity5822 expression using flow cytometry. Contamination by mesenchymal cells, extravillous trophoblast cells, leukocytes, Hofbauer and endothelial cells is minimized (less than 2  % when analyzed for @entity2277 , @entity6393 , @entity1195 , @entity11772 and @entity2279 using flow cytometry). Isolated cytotrophoblast cells began to aggregate into monolayers of mononucleated cells within about 12  h of plating. By 72  h in culture, most cytotrophoblast cells have differentiated into syncytiotrophoblast as demonstrated by a loss of intercellular @entity5962 expression upon fusion into multinucleated syncytia. After 72  h in culture, nearly every cultured cell expresses syncytiotrophoblast markers, including @entity5822 , @entity1 chorionic gonadotropin-b (b-hCG) and the fusion-related proteins @entity2430 ( @entity2430 ) and syncytin. CONCLUSIONS: We present an efficient and reliable method for isolating of cytotrophoblast cells with high purity and complete differentiation into syncytiotrophoblast in vitro.
