Stem cells and their progeny, from all ages of the CNS, can be stimulated to proliferate when they are exposed to growth factors in tissue culture1. When appropriate plating densities are established in culture, continued cell division generates non-adherent spherical clusters of cells, commonly referred to as neurospheres2,3. The neurosphere assay has proven to be an excellent technique to isolate neural stem cells and progenitor cells to investigate the differentiation and potential of cell lineages. These spheres can be dissociated, expanded and pooled in sufficient quantity for subsequent scientific inquiry. With many potential therapeutic applications for neural stem cells4, qualitative and quantitative insight into the precise cellular makeup of the neurosphere is required.
Many factors contribute to the cellular composition of the neurosphere. Key variables including the age of the animal, plating density and passage number will all influence the homogeneity or heterogeneity of the neurosphere5,6. While the neurosphere assay is a valuable procedure from many biological perspectives, the user should be aware that there are particular relationships between the definitive neural stem cell and the production of neurospheres in culture that the can lead to shortcomings7.
We describe the steps in detail to isolate and expand neural stem cells in the form of neurospheres from tissue dissections of the post-natal mouse brain. Procedures for the long term passage of neurospheres and the cryopreservation of neurospheres are also provided. In addition to the guidelines and tips for generating neurosphere cultures, we describe the method to prepare neurospheres for analysis by light microscopy. The ability to section neurospheres for histology or immunocytochemistry provides the researcher with the additional dimension to study specific molecular and cellular aspects of the neurosphere. Many of the commercial kits designed to assess aspects of the cell cycle, programmed cell death, and cell signaling can be readily applied to the outlined protocols.