We have prepared highly purified cultures of rat oligodendrocytes by a modification of the procedure of McCarthy and de Vellis [1980]. By utilizing a substratum derived from lysed glia and a calf serum-containing medium with a high concentration of transferrin, the oligodendrocyte cultures display a high degree of purity, the ability to survive several months of culture, and a striking ability to produce a myelin-like membrane. We have examined the production of this myelin-like membrane using immunocytochemical and biochemical probes as well as an extensive morphological examination at the electron microscopic level. The membrane appears to be produced in a similar developmental pattern to that observed in vivo and it has the structural characteristics of loosely packed central nervous system myelin.

Myelin deposition and maintenance are critical to proper function of the mammalian nervous system. Previous investigations of myelination in the central nervous system (CNS) were hampered by the lack of an in vitro system that can faithfully reproduce in vivo events yet is amenable to biochemical investigation. We have developed a procedure, based on organotypic cultures, which permits efficient preparation of large numbers of cerebellar slice cultures that can be easily manipulated. Cultures have been examined to document myelination biochemically (by incorporation of [35S]sulfate into sulfolipids), immunohistochemically (by labeling the myelin components myelin basic protein and galactocerebroside), and morphologically (by both light and electron microscopy). We tested the effects of biologically active peptides and antibodies on myelination in the thin slices. The results indicate that the cultures provide an in vitro system that can be used to examine specific cellular events that occur during CNS myelination.

A system was developed to analyze early events in the process of myelination. Primary cultures of rat oligodendrocytes were maintained in the presence of glass micro-fibers which served as artificial axons. A culture chamber was constructed which allowed the close apposition of fibers and cells in a three-dimensional arrangement designed to resemble an in vivo environment. Cells cultured in the presence of glass micro-fibers coated with a glial cell matrix extract were induced to organize into clusters around the fibers. Examination of oligodendrocyte-fiber sandwiches by SEM revealed the presence of a number of cell contacts with the fibers. TEM images showed that, in most cases, fibers were surrounded by the cells and not multiply wrapped. Only occasionally was a loose wrapping of cell membrane observed around the fibers. Cells cultured in the presence of matrix-coated glass micro-fibers showed an increased production of sulfolipids that was at least partially dependent on the presence of the matrix coating. Coating of these "artificial axons" may aid in the identification of signal molecules produced by neurons which enable them to be myelinated.

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