Neural Stem Cell Culture: Neurosphere generation, microscopical analysis and cryopreservation

Abstract

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.