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Formation of Bovine Placental Trophoblast Spheroids

Introduction: In this study, we aimed to form spheroids with the bovine placental trophoblast cell line F3. Spheroids are 3-dimensional culture models which can be used to conduct versatile in vitro and in vivo experiments. Materials and Methods: The spheroids were generated using the hanging drop technique, 25% methocel and matrigel. The F3 spheroids were characterized morphologically by light microscopy and transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemistry (ezrin, vimentin, cytokeratin, placental lactogen). The fluorescent dyes calcein and ethidium homodimer were used to determine the viability of the spheroidal F3 cells by immunofluorescence microscopy. Results: The cell line F3 only formed spheroids by the hanging drop technique when matrigel was added. The trophoblast spheroids were delimited and fully covered by extracellular matrix (light microscopy/TEM/SEM). Cells contributing to spheroids could not be discriminated from each other (light microscopy). The outer spheroidal layer consisted of cells which possessed an apical pole with microvilli that were directed to the outside (light microscopy/TEM). All of the spheroidal F3 cells expressed ezrin, vimentin and cytokeratin, but not placental lactogen. The spheroid core contained degenerating cells whilst the F3 cells of the outer rim were viable (TEM/immunofluorescence microscopy). Discussion: We have established a 3-dimensional spheroid model for the bovine placental trophoblast cell line F3. The developed culture model might prove valuable for future in vitro studies on the differentiation of bovine trophoblast cells.