Passaging protocols for mammalian neural stem cells in suspension bioreactors

Mammalian neural stem cells (NSC) offer great promise as therapeutic agents for the treatment of central nervous system disorders. As a consequence of the large numbers of cells that will be needed for drug testing and transplantation studies, it is necessary to develop protocols for the large-scale expansion of mammalian NSC. Neural stem cells and early progenitor cells can be expanded in vitro as aggregates in controlled bioreactors using carefully designed media. The first objective of this study was to determine if it is possible to maintain a population of murine neural stem and progenitor cells as aggregates in suspension culture bioreactors over extended periods of time. We discovered that serial passaging of a mixture of aggregates sizes resulted in high viabilities, high viable cell densities, and good control of aggregate diameter. When the NSC aggregates were serially subcultured three times without mechanical dissociation, a total multiplication ratio of 2.9 x 10(3) was achieved over a period of 12 days, whereas the aggregate size was controlled (mean diameter less than 150 microm) below levels at which necrosis would occur. Moreover, cell densities of 1.0 x 10(6) cells/mL were repeatedly achieved in batch culture with viabilities exceeding 80%. The second objective was to examine the proliferative potential of single cells shed from the surface of these aggregates. We found that the single cells, when subcultured, retained the capacity to generate new aggregates, gave rise to cultures with high viable cell densities and were able to differentiate into all of the primary cell phenotypes in the central nervous system.