Isolation and characterization of murine retinal endothelial cells.
OBJECTIVE
To isolate and characterize primary retinal endothelial cells (REC) from wild type and transgenic mice to facilitate the study of their properties in vitro.


METHODS
REC were isolated from wild type or transgenic-immortomouse by collagenase digestion of retina and affinity purification using magnetic beads coated with platelet/endothelial cell adhesion molecule-1 (anti-PECAM-1). The bound cells were plated on fibronectin-coated wells and expanded. The REC were characterized for expression and localization of endothelial cell markers by fluorescence-activated cell sorting (FACS) analysis and indirect immunofluorescence staining. The ability of these cells to form capillary like networks was assessed on Matrigel while the migration properties were examined in wound closure assays.


RESULTS
Isolation of REC from mouse has been very difficult and has not been previously reported. Here, we describe a method for isolation of retinal endothelial cells from wild type and thrombospondin-1 deficient (TSP1-/-) immortomice. Our results indicate that nearly 100% of selected cells express the endothelial cell marker PECAM-1 and vascular endothelial-cadherin (VE-cadherin). The cells were successfully passaged and maintained in culture for several months without a significant loss in expression of endothelial cell markers. The wild type REC, like most primary endothelial cells, formed capillary-like networks on Matrigel. The ability of the REC from TSP1-/- mice to form capillary-like networks on Matrigel was severely compromised. This may be attributed, at least in part, to the enhanced migratory and less differentiated phenotype of these cells.


CONCLUSIONS
The retinal endothelial cells can be readily obtained from wild type and transgenic mice, which facilitate the comparison and identification of the physiologic role of specific genes in endothelial cell function.
Department of Ophthalmology & Visual Sciences, University of Wisconsin, Madison 53792-4673, USA.
Xiaojing
X
Su
Christine M
CM
Sorenson
Nader
N
Sheibani
D000818:Animals; D015703:Antigens, CD; D015153:Blotting, Western; D015820:Cadherins / Q000378:metabolism; D002196:Capillaries / Q000166:cytology; D018929:Cell Culture Techniques / Q000379:methods; D002465:Cell Movement / Q000502:physiology; D002469:Cell Separation / Q000379:methods*; D002846:Chromatography, Affinity; D004730:Endothelium, Vascular / Q000166:cytology* / Q000502:physiology; D005353:Fibronectins / Q000378:metabolism; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D018189:Immunomagnetic Separation; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D018919:Neovascularization, Physiologic / Q000502:physiology; D010750:Phosphoproteins / Q000378:metabolism; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism; D012171:Retinal Vessels / Q000166:cytology*; D019700:Thrombospondin 1 / Q000172:deficiency / Q000235:genetics / Q000378:metabolism; D062826:Zonula Occludens-1 Protein; D000099152:Cadherin 5
D015703:Antigens, CD; D015820:Cadherins; D005353:Fibronectins; D008565:Membrane Proteins; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D019700:Thrombospondin 1; D062826:Zonula Occludens-1 Protein; D000099152:Cadherin 5; C569043:Tjp1 protein, mouse