Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1

Takashi Matsubara Department of Ophthalmology, Kansai Medical University, Osaka, Japan; Children’s Hospital/Harvard Medical School, Boston, Massachusetts; Doheny Eye Institute and Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles, California 90033

Toshinori Murata Department of Ophthalmology, Kansai Medical University, Osaka, Japan; Children’s Hospital/Harvard Medical School, Boston, Massachusetts; Doheny Eye Institute and Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles, California 90033

Guey-Shuang Wu Department of Ophthalmology, Kansai Medical University, Osaka, Japan; Children’s Hospital/Harvard Medical School, Boston, Massachusetts; Doheny Eye Institute and Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles, California 90033

Ernesto Barron Department of Ophthalmology, Kansai Medical University, Osaka, Japan; Children’s Hospital/Harvard Medical School, Boston, Massachusetts; Doheny Eye Institute and Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles, California 90033

Narsing A. Rao Department of Ophthalmology, Kansai Medical University, Osaka, Japan; Children’s Hospital/Harvard Medical School, Boston, Massachusetts; Doheny Eye Institute and Department of Ophthalmology, University of Southern California, School of Medicine, Los Angeles, California 90033

Abstract

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.

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Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1

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Isolation and culture of rat retinal microvessel endothelial cells using magnetic beads coated with antibodies to PECAM-1

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