title: Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1.
abstract: OBJECTIVE
  To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to rat platelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs.
  
  
  METHODS
  Magnetic beads were coated with anti-rat PECAM-1 antibodies. Retinas were obtained from Lewis rat eyes. After the retinas were digested with collagenase, they were incubated with the antibody-coated beads with agitation. RECs that stuck to the beads were collected with a magnetic particle concentrator and cultured in fibronectin coated wells. The characteristics of the RECs were examined by immunohistochemical study utilizing von Willebrand's Factor, acetylated low-density lipoprotein uptake and transmission electron microscopy.
  
  
  RESULTS
  The cells isolated using the PECAM-1-coated magnetic-bead technique formed a contact-inhibited cobblestone monolayer that stained positive for von Willebrand's Factor. These cells revealed low-density lipoprotein uptake. Ultrastructurally, the isolated cells exhibited pinocytic vesicles and a high density of intercellular junctions without fenestrations.
  
  
  CONCLUSIONS
  These results indicate that the isolated cells were vascular endothelial cells showing both morphologic and functional characteristics of retinal vascular endothelium. The magnetic-bead technique was useful for isolating high purity RECs that can be cultured to study the physiological, immunological and biochemical role of the endothelium in various ocular diseases.
authors.affiliation: Department of Ophthalmology, Kansai Medical University, Osaka, Japan.
authors.forename: T A
authors.initials: TA
authors.lastname: Matsubara
authors.forename: T A
authors.initials: TA
authors.lastname: Murata
authors.forename: G S
authors.initials: GS
authors.lastname: Wu
authors.forename: E A
authors.initials: EA
authors.lastname: Barron
authors.forename: N A
authors.initials: NA
authors.lastname: Rao
mesh_terms: D000818:Animals; D000906:Antibodies; D002232:Carbocyanines / Q000493:pharmacokinetics; D002469:Cell Separation / Q000379:methods*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D008077:Lipoproteins, LDL / Q000378:metabolism; D008280:Magnetics; D008863:Microspheres; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000276:immunology; D051381:Rats; D011917:Rats, Inbred Lew; D012171:Retinal Vessels / Q000166:cytology* / Q000378:metabolism / Q000648:ultrastructure; D014841:von Willebrand Factor / Q000378:metabolism
chemical_list: D000906:Antibodies; D002232:Carbocyanines; D005456:Fluorescent Dyes; D008077:Lipoproteins, LDL; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C032629:acetyl-LDL; D014841:von Willebrand Factor; C024286:3,3'-dioctadecylindocarbocyanine