Binding of human endothelium to Ulex europaeus I-coated Dynabeads: application to the isolation of microvascular endothelium.
A major problem encountered when isolating human microvascular endothelium is the presence of contaminating cells such as fibroblasts that rapidly over-grow the endothelial cells. We describe here a simple, rapid technique for purifying endothelial cells derived from the microvasculature of neonatal foreskin and osteoarthritic and rheumatoid arthritic synovium. This technique is based on the selective binding of the lectin Ulex europaeus I (UEA I) to the endothelial cell surface via fucose residues. Initially UEA I was covalently bound to tosyl-activated super-paramagnetic polystyrene beads (Dynabeads) by incubation for 24 h at room temperature. Cells were isolated by extracting microvascular segments from enzyme-treated (trypsin and Pronase) cubes of tissue. The mixed population of cells obtained were purified by incubating them at 4 degrees C for 10 min with the UEA I-coated Dynabeads. Endothelium bound to the beads whilst contaminating cells were removed by five washes with HBSS using a magnetic particle concentrator. The endothelial cells thus obtained grew to confluence as a cobblestone-like monolayer and expressed von Willebrand factor antigen. The cells were released from the Dynabeads by the competitive binding of fucose (10 min at 4 degrees C). This new method is simple and reproducible and allows pure human microvascular endothelial cells to be cultured within 2 h of obtaining a specimen.
Jackson
C J
CJ
Sydney University Department of Rheumatology, Royal North Shore Hospital, St Leonards, NSW, Australia.
Garbett
P K
PK
Nissen
B
B
Schrieber
L
L
D002460:Cell Line; D002469:Cell Separation / Q000379:methods*; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005260:Female; D005455:Fluorescent Antibody Technique; D005643:Fucose / Q000378:metabolism; D006801:Humans; D037102:Lectins / Q000378:metabolism*; D008297:Male; D008863:Microspheres; D037121:Plant Lectins; D014841:von Willebrand Factor / Q000378:metabolism
D037102:Lectins; D037121:Plant Lectins; C036850:Ulex europaeus lectins; D014841:von Willebrand Factor; D005643:Fucose