AbstractA subset of oligodendrocytes (B3,f) was isolated by taking advantage of selective cell–substratum interaction. B3,fcells were characterized morphologically, biochemically, and immunocytochemically. Oligodendrocytes were isolated from 4‐to‐6‐month‐old lamb brains by a modified version of our published procedure [Szuchet et al, J Neurosci Methods 3:7–19, 1980]. Freshly isolated cells from band III were plated on plastic culture plates at a concentration of 2 × 106cells/ml. Approximately 40% of the cells attached to the plate under these conditions. The remaining cells formed small floating clusters. We refer to the latter as B3,foligodendrocytes. After 4 to 5 days, the supernatant containing B3,fcells was removed and centrifuged, and the pellet was resuspended in culture medium and replated on polylysine‐coated petri dishes. B3,foligodendrocytes attached to this surface and extended an intricate network of processes. The purity of the cultures, judged by the number of cells staining with a monoclonal antibody against galactocerebroside was 98–99%. This high degree of cell homogeneity was maintained throughout the life of the cultures. B3,fcells appeared to be highly differentiated and remained so in vitro. This is surmised by the expression of oligodendrocytic characteristic functions such as high levels of CNPase activity typically, 5 μM/min/mgP; high incorporation of H235SO4into sulfatides, an overall lipid metabolism that mimics events associated with myelinogenesis [Szuchet et al, PNAS 80:7019–7023, 1983]; the presence, detected immunocytochemically, of myelin‐associated glycoprotein and myelin basic proteins. It is concluded that this culture system offers an opportunity for studying the biology of interfascicular oligodendrocytes and their interaction with neurons and/or astrocytes. It also should open up a way of examining the relevance of oligode
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