Isolation and characterization of murine retinal astrocytes.
OBJECTIVE
To isolate and characterize primary retinal astrocytes in culture (RAC) from wild-type and transgenic mice to aid the study of their properties in vitro.


METHODS
Astrocytes were isolated from wild-type and transgenic Immortomice by collagenase digestion of the retina. Affinity purification using magnetic beads coated with anti-platelet endothelial cell adhesion molecule-1 (PECAM-1) was used to remove retinal endothelial cells. The remaining cells were cultured and expanded. The majority of these cells were identified as astrocytes. These cells were characterized for expression of astrocytic markers using fluorescence-activated cell sorting (FACS) and immunostaining analysis. The expression of various integrins and other cell adhesion molecules on the surface of retinal astrocytes, their adhesion to various matrix proteins, their migration, and their ability to organize on Matrigel were determined.


RESULTS
Here we describe a method for the isolation of RAC from wild-type and thrombospondin-1 deficient (TSP1-/-) mice. Our results indicated that nearly 100% of cells isolated expressed the astrocytic markers GFAP, NG2, Pax2, and vimentin. These cells were successfully passaged and maintained in culture for several months without a significant loss in expression of astrocytic markers. The RAC expressed alphavbeta3 integrin and other cell adhesion molecules on their surface. The TSP1-/- RAC adhered more strongly to fibronectin and vitronectin compared to the wild-type cells, while neither cell types adhered to collagen and laminin. Wild-type and TSP1-/- RAC exhibited similar migratory characteristics despite alterations in their adhesive properties and production of various matrix proteins. Also, these cells, like endothelial cells, similarly organized into a network in Matrigel.


CONCLUSIONS
The RAC can be readily obtained from wild-type and transgenic mice. This facilitates the comparison and identification of specific gene functions in RAC compared to astrocytes prepared from other sites of central nervous system.
Scheef
Elizabeth
E
Department of Ophthalmology & Visual Sciences, University of Wisconsin Medical School, Madison, WI 53792-4673, USA.
Wang
Shoujian
S
Sorenson
Christine M
CM
Sheibani
Nader
N
D000818:Animals; D000941:Antigens / Q000378:metabolism; D001253:Astrocytes / Q000166:cytology* / Q000378:metabolism; D018929:Cell Culture Techniques; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D005904:Glial Fibrillary Acidic Protein / Q000378:metabolism; D018189:Immunomagnetic Separation; D039302:Integrin alphaVbeta3 / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D051762:PAX2 Transcription Factor / Q000378:metabolism; D011509:Proteoglycans / Q000378:metabolism; D012160:Retina / Q000166:cytology* / Q000378:metabolism; D019700:Thrombospondin 1 / Q000172:deficiency; D014746:Vimentin / Q000378:metabolism; D000099216:Chondroitin Sulfate Proteoglycan 4
D000941:Antigens; D005904:Glial Fibrillary Acidic Protein; D039302:Integrin alphaVbeta3; D051762:PAX2 Transcription Factor; D011509:Proteoglycans; D019700:Thrombospondin 1; D014746:Vimentin; D000099216:Chondroitin Sulfate Proteoglycan 4; C497319:Pax2 protein, mouse