In vivo studies of myelination and demyelination offer only snapshot views of these dynamic processes and do not permit longitudinal study at the cellular level, whilst 2D cultures fail to reproduce many of the most basic features of the complex spatial and mechanical environment in which CNS myelination occurs. We are developing a 3D myelinating cell culture model that is amenable to continuous observation and controlled manipulation, which will allow us to follow the dynamic process of myelination in vitro. Oligodendrocyte precursor cells (OPCs) are isolated from P1-2 rat pups, seeded into poly-L-ornithine-coated flasks and expanded by treatment with b-FGF and PDGF. OPCs are recovered and suspended within a neutralized solution of Type-I rat-tail collagen. This suspension is then poured into wells of a 24-well or 96-well plate, where it sets to form a gel with cells seeded evenly throughout its volume. Gels are 1 to 2 mm thick; sufficient to provide a 3D environment for the cells, yet still permit microscopic monitoring at all depths. Differentiation is triggered by withdrawal of PDGF and bFGF and addition of medium containing triiodothronine, ciliary neurotrophin factor (CNTF) and N-acetyl cysteine. Within 2 days, expression of MBP is observed in differentiating oligodendrocytes, and, in co-cultures with neurones, MBP1 oligodendrocytes are observed associated with axonal processes. Electron microscopic analysis of cultures that were maintained in differentiation medium for 3 days revealed myelin-like structures. Unlike existing 3D in vitro systems such as CNS explant cultures or reaggregate CNS cultures, our new system permits live cell imaging and time-lapse microscopy during myelination. We are developing our model further to include other CNS cell populations and to further optimise myelin formation. This culture system has unique potential to study the dynamics of demyelination and to explore the potential of therapies to enhance remyelination.