Isolation, culture, and characterization of adult rat oligodendrocytes

Abstract

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 (BSA). The pellet was resuspended and placed in an untreated 6-well culture dish overnight to allow the astrocytes to attach. The non-adherent cells were replated on poly-l-lysine-treated coverslips. Approximately 8.25 x 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 membrane sheath between these processes was immunostainable with either anti-galactocerebroside (GC), anti-O4, anti-myelin basic protein (MBP), or anti-2'3' cyclic nucleotide 3' phosphohydrolase (CNPase) and was also evident in scanning EM. Older cultures (up to 60 DIV) maintained whorls of myelin and transmission EM revealed a major dense line distance of approximately 103 A with up to 11 concentric layers of membrane. Immunologically, the adult oligodendrocytes are GC+, O4+, 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.