Human capillary endothelial cells from abdominal wall adipose tissue: isolation using an anti-pecam antibody.
We have developed a novel isolation technique for harvesting human capillary endothelial cells. We compared the use of either Ulex Europaeus Agglutinin (UEA) lectin or anti-platelet endothelial cell adhesion molecule (PECAM) antibody conjugated to magnetic beads for the ability to isolate and maintain pure cultures of human capillary endothelial cells. Cells isolated using either method actively scavenged DiI-acetylated-low density lipoprotein and expressed von Willebrand factor (vWf) up to four passages as assessed by immunofluorescent labeling. Endothelial cells isolated using the anti-PECAM antibody method maintained these endothelial-specific properties for up to 12 passages while the percentage of UEA selected cells expressing these properties decreased during increasing passage number. Furthermore, while both techniques yielded cells that bind UEA at Passage six, only the antibody selected cells expressed the normal pattern of endothelial-specific cellular adhesion molecules as assessed by flow cytometry. Both cell isolates were cultured within a three-dimensional matrix of type I collagen, the antibody selected cells formed tubelike structures within 2 days, while the lectin selected cells did not. The antibody selected capillary endothelial cells were transduced with a retroviral vector containing the human growth hormone cDNA and were found to secrete growth hormone from both two- and three-dimensional cultures. We propose that anti-PECAM antibodies linked to a solid support provide a highly selective step in the isolation and maintenance of pure populations of human capillary endothelial cells from abdominal wall liposuction remnants.
Springhorn
J P
JP
Department of Molecular Development, Alexion Pharmaceuticals, Inc., New Haven, Connecticut 06511, USA.
Madri
J A
JA
Squinto
S P
SP
D000005:Abdomen; D000273:Adipose Tissue / Q000166:cytology*; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D015214:Antigens, Differentiation, Myelomonocytic / Q000276:immunology*; D015815:Cell Adhesion Molecules / Q000276:immunology*; D002469:Cell Separation / Q000379:methods*; D002472:Cell Transformation, Viral; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology*; D013006:Growth Hormone / Q000096:biosynthesis; D006801:Humans; D037102:Lectins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019010:Vascular Cell Adhesion Molecule-1 / Q000096:biosynthesis; D014841:von Willebrand Factor / Q000096:biosynthesis
D000911:Antibodies, Monoclonal; D015214:Antigens, Differentiation, Myelomonocytic; D015815:Cell Adhesion Molecules; D037102:Lectins; D037121:Plant Lectins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C036850:Ulex europaeus lectins; D019010:Vascular Cell Adhesion Molecule-1; D014841:von Willebrand Factor; D013006:Growth Hormone