PURPOSE: 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. CONCLUSION: 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.
PURPOSE: To isolate retinal endothelial cells (RECs) from Lewis rats using magnetic beads coated with antibodies to ratplatelet-endothelial cell adhesion molecule-1 (PECAM-1) and to characterize the cultured RECs. METHODS: Magnetic beads were coated with anti-ratPECAM-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. CONCLUSION: 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: William M Watkins; Gary W McCollum; Sara R Savage; Megan E Capozzi; John S Penn; David G Morrison Journal: Exp Eye Res Date: 2013-09-26 Impact factor: 3.467
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Authors: Melane L Fehrenbach; Gaoyuan Cao; James T Williams; Jeffrey M Finklestein; Horace M Delisser Journal: Am J Physiol Lung Cell Mol Physiol Date: 2009-03-20 Impact factor: 5.464