Culture of endothelial cells from human placental microvessels

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 alpha-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.

Phenotype of cultured fetal perivascular cells from human placenta studied by scanning electron microscopy

“…These circular structures were also observed in brain MEC cultured on Matrigel (Gerhart et al 1988). The MEC were stained by the von Willebrand factor reaction and took up fluorescent acetylated low-density lipoproteins (Kacemi et al 1996). There was no evidence for contaminating cells exhibiting a pericyte phenotype or actin stress fibers as we have previously described using the placenta (Kacemi et al 1996) or as shown by others with the brain (Rupnick et al 1988;Abbott et al 1992).…”

“…The MEC were stained by the von Willebrand factor reaction and took up fluorescent acetylated low-density lipoproteins (Kacemi et al 1996). There was no evidence for contaminating cells exhibiting a pericyte phenotype or actin stress fibers as we have previously described using the placenta (Kacemi et al 1996) or as shown by others with the brain (Rupnick et al 1988;Abbott et al 1992). MEC might modify their phenotype according to the matrix components such as collagen, laminin (Kubota et al 1988) or Matrigel (Kubota et al 1988Gerhart et al 1988).…”

“…Four to five placental lobes were immediately excised. The isolation and culture of endothelial cells from placental microvessels has previously been described (Kacemi et al 1996). Briefly, villi were sized by sieving (75-µm mesh) and digested twice with a mixture of 0.1% collagenase-dispase (Boehringer) and 20 U/ml DNAse (Type I, Sigma, St. Quentin Fallavier, Fance) for 2 h at 37°C in a shaking incubator (100 cycles/min).…”

“…Confluence was reached after 7-10 days. These confluent cultures were incubated with microbeads (M450, Dynal, Compiègne, France) coated with human 24FM antithrombomodulin (Serbio, Gennevilliers, France) as previously described (Kacemi et al 1996), washed and subcultured on gelatinized coverslips.…”

“…Many studies Gebrane-Younes et al 1986) have described the perivascular cell types and their distribution and density in the umbilical-placental circulation, but their phenotype has mostly been evaluated from tissue sections. Moreover, except for human endothelial cells from the umbilical vein (HUVEC), the phenotype of these perivascular cells has been poorly studied outside their normal constraints (Leach et al 1994;Challier et al 1995;Kacemi et al 1996).…”