Functional, long-term cultures of human term trophoblasts purified by column-elimination of CD9 expressing cells

We have extended previous observations of expression of the trypsin-resistant cell surface antigen CD9 on placental fibroblasts to virtually all cells in the villous stroma and developed a method for eliminating CD9 expressing cells from trypsinized placental preparations. Preparations incubated with the mouse anti-human CD9 monoclonal antibody 50H.19 were passed through a goat anti-mouse immunoglobulin column that captures CD9 expressing cells. Approximately 95 per cent of the eluted cells stained positive with the villous trophoblast specific antibody GB25 and could be cryopreserved and thawed with > 80 per cent recovery in culture. One week cultures contained fewer than 0.3 per cent vimentin positive (mesenchymal) cells and maintained secretion of hCG. Two week cultures remained free of fibroblasts and macrophages. Clusters of trophoblasts that formed spontaneously during the first week of culture were shown by microinjection of carboxyfluorescein and by staining with anti-desmoplakin antibody to be a patchwork of mononuclear cells and syncytial units. Although the DNA content of the culture decreased by 35 per cent during the 2 week culture, the metabolic capacity and protein content remained relatively constant. Thus, CD9 immuno-elimination gives a high yield of enriched and viable trophoblasts that can be cultured for at least 2 weeks with almost no contamination by stromal cells.