Culture of endothelial cells from human placental microvessels

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Abstract
Endothelial cells are known to participate in angiogenesis, adaptation of vascular tonus and maintenance of blood fluidity in the microcirculation. To investigate these functions in the placenta, we devised a method of isolation and culture of endothelial cells from villous microvessels. In primary culture, these intraplacental endothelial cells exhibited many features observed in microvascular endothelium from other organs: spindle-shape, rosette associations, circular arrangements and confluence. In contrast to the confluent endothelial cells derived from the umbilical vein, cells from microvessels did not form a cobblestone network. After trypsin digestion of microvessels, magnetic microbeads coated with S-Endol immunoglobulin, antithrombomodulin and Ulex europaeus-I lectins were tested for sorting endothelial cells. Only the microbeads coated with antithrombomodulin allowed a suitable magnetic cell separation after trypsinization. By contrast, the microbeads coated with each of these antibodies or with lectins attached to confluent cells from the second passage. The microbeads detached from the cells at different rates. Their examination by scanning microscopy indicates that a portion of these microbeads was phagocytosed. Microvascular endothelial cells from the second passage were intensively stained by the anti-von Willebrand reaction and only weakly by the anti-smooth muscle α-actin reaction. They incorporated acetylated-low density lipoproteins coupled to a fluorescent probe. The positive reactions against the anti-von Willebrand factor and the uptake of the fluorescent acetylated-low density lipoproteins were modified after eight passages.

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