Neurosphere Cultures

The Neurosphere Method

The neurosphere system can be applied to many different tissues, be they of adult, fetal, or embryonic origin, from virtually all mammalian species, including humans. In its most common application, [Page 378]donor tissue is predigested enzimatically and then mechanically dissociated to yield a single—cell suspension, which is then plated under quite stringent growth conditions. This procedure establishes a selective culture system in which most of the primary differentiated/differentiating central nervous system cells found in the primary tissue die out soon after plating, whereas the undifferentiated stem cells enter into a state of active proliferation.

The discovery that some regions of the mature brain are the site of intense neurogenesis throughout life has changed some of our understanding of the brain. The biological entity at the root of this process is the neural stem cell.

Four main conditions must absolutely be satisfied for the neural stem cells to become the prevalent cell type in these cultures: there must be low cell density (<5 × 104 cells/cm2), there must be an absence of serum, and there must be the addition of the appropriate growth factors (i.e., EGF or FGF2), and there must be a plating substrate warranting loose cell adhesion (poly—L-lysine or poly—ornithine may be used). Under these conditions, cells from a freshly dissociated brain attach loosely to the substrate, with the majority (99 percent) rapidly dying out. At the same time, a tiny fraction of undifferentiated neural precursors, mostly neural stem cells, become hypertrophie, round up, and engage into active proliferation while adhering loosely to the culture vessel. The progeny of these proliferating precursors preferentially adhere to each other while dividing and, eventually, form spherical clusters that, because of their increasing mass, eventually lift off the substrate and float in suspension. These have been named neurospberes, from which comes the name of the technique.

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