Murine spinal cord explants: a model for evaluating axonal growth and myelination in vitro

In vitro models of myelinating central nervous system axons have mainly been of two types, organotypic or dissociated. In organotypic cultures, the tissue fragment is thick and usually requires sectioning (physically or optically) before visual examination. In dissociated cultures, tissue is dispersed across the culture surface, making it difficult to measure the extent of myelinated fiber growth. We aimed to develop a method of culturing myelinated CNS fibers in defined medium that could be 1) studied by standard immunofluorescence microscopy (i.e., monolayer type culture), 2) used to measure axonal growth, and 3) used to evaluate the effect of substrate and media components on axonal growth and myelination. We used 120-micro m slices of embryonic murine spinal cord as a focal source of CNS tissue from which myelinated axons could extend in a virtual monolayer. Explants were cultured on both poly-L-lysine and astrocytes. The latter were used because they are the scaffold on which axonal growth and myelination occurs during normal development. Outgrowth from the explant and myelination of axons was poor on poly-L-lysine but was promoted by an astrocyte bed layer. The best myelin formation occurred in defined media based on DMEM using N2 mix; it was not promoted by Sato mix or Neurobasal medium with B27 supplement. Neuronal survival was poor in serum-containing medium. This tissue culture model should facilitate the study of factors involved in promoting outgrowth of CNS axons and their myelination. As such it is relevant to studies on myelination and spinal cord repair.