Isolation, culture, and characterization of adult rat oligodendrocytes

Investigation into CNS demyelinating diseases, which usually occur in adults, can be facilitated by the use of a good in vitro model. We have established a methodology whereby oligodendrocytes from adult rat CNS can be cultured in vitro, and we have characterized these cultures morphologically and immunologically. Approximately 1 g of spinal cord and brainstem per adult rat was removed and dissociated mechanically and enzymatically. After filtration of the white matter homogenate, myelin was removed by 0.9 M sucrose density centrifugation. The cells were further purified by centrifugation through a 0.3%/4% discontinuous gradient of bovine serum albumin (BSAZ). The pellet was resuspended and placed in an untreated 6‐well culture dish overnight to allowthe astrocytes to attach. The non‐adherent cells were replated on poly‐l‐lysine‐treated coverslips. Approximately 8.25 × 10 5 cells were recovered per animal. The adult oligodendrocytes initially appeared as rounded cell bodies, but after 2–5 days in vitro (DIV), the oligodendrocytes extended 6–10 thick processes. A membrance sheath between these processes was immunostainable with either anti‐galactocerebroside (GC), anti‐04, anti‐myelin basic protein (MBP), or anti‐2′3′ cyclic nucleotide 3′ phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up nto 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 AS with up to 11 concentric layers of membrance. Immunologically, the adult oligodendrocytes are GC + , 04 + , MBP + , CNPase + , and GFAP − . The method described will allow adult rat oligodendrocytes to be isolated and maintained in culture; these cultures retain the characteristics of differentiated adult oligodendrocytes.