Mixed culture of pericytes and endothelial cells from fetal microvessels of the human placenta.

To explore the role of perivascular cells in angiogenesis and vasomotricity, placental cultures of perivascular cells were performed from calibrated villi excised from term placentas. Microvessels were isolated using repeated digestion of villi by collagenase-dispase and purification by Percoll gradients. Plated on Petri dishes, the microvessels became adherent to the gelatin matrix permitting to cells to proliferate. Cells were harvested and subcultured. Endothelial and pericyte cell lines were identified by phase contrast microscopy. Pericyte number predominated rapidly, the endothelial cells remaining visible. After seven days, cells started to cluster, thus piled up and built numerous nodules. Medium-size oval endothelial cells were stained by anti-von Willebrand factor and anti-IgG coupled to fluorescein. Large cells with irregular border reacted to smooth muscle anti-alpha-actin and anti-IgG coupled to fluorescein. There was no cross-reaction of these two cell types with the antibodies. In contrast, nodules were stained by both immunostainings. Endothelial cells reacting to von Willebrand factor antibody were frequently associated to the nodule. The isolation of microvessels from the human placenta described in this study allowed the establishment of cultures of endothelial cells and pericytes that show: i) rapid predominance of pericytes over endothelial cells, ii) formation of nodules, iii) participation of endothelial cells and pericytes to nodules formation.