A procedure has been developed which yields a pure population of villous cytotrophoblast from term human placenta. As a first step, a cell preparation highly enriched for cytotrophoblast (identified by positive cytokeratin staining) was obtained using a modification of the method of Kliman et al. (1986). The remaining contaminating cells (identified by positive vimentin staining) were then removed by treatment with mouse monoclonal antibodies against class I and class II major histocompatibility antigens followed by magnetic microspheres coated with goat anti-mouse IgG. The rationale for this step was based on the fact that villous trophoblast fails to express HLA antigens whereas cells from the villous mesenchyme do express these surface antigens. Rosetted cells were immobilized using a magnet allowing the non-rosetted cells to be easily withdrawn by pipette. When the non-rosetted cells were placed in primary culture, no HLA-positive or vimentin-positive cells could be detected using immunofluorescence microscopy, indicating complete removal of these components by the immunomagnetic separation procedure. The cells were positive for cytokeratin and, after 24 h, showed positive staining for pregnancy-specific β1-glycoprotein (SP1) and human chorionic gonadotropin. Recovery of cytotrophoblast was greater than 92% with only a slight loss of viability.
- Magnetic microsphere
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