Neural stem cells (NSC) represent a potential source of non-transformed cells for drug testing and transplantation for the treatment of such diseases as Parkinson's, Huntington's and multiple sclerosis. As a result, there is an enormous demand to develop methods for the expansion of these cells. Consequently, the aim of this study is to develop bioreactor protocols for the large-scale growth of mammalian neural stem cells as aggregates.

In this study, it is proven that mammalian NSC can successfully be expanded in suspension culture. Many issues regarding the suspension culture growth of mammalian NSC are identified and examined in detail. These include inoculation protocols, expansion protocols, key environmental conditions, passaging techniques, nutrient and oxygen uptake, and cell cycle kinetics. Manipulation of the shear field and the addition of media additives helped to maintain the average spheroid diameter below sizes that would cause necrotic centers to develop. Moreover, murine NSC were successfully serially passaged in suspension culture for over 35 days with an overall multiplication ratio of over 107 and no discernable loss in growth potential or stem cell attributes. The cells remained karyotypically normal and retained their ability to be differentiated into all three major cell types of the central nervous system (CNS) - neurons, astrocytes, and oligodendrocytes. Finally, the growth of human NSC in stationary and suspension culture was also successfully accomplished, with both fed-batch and passaging techniques examined. This represents a major milestone on the road to effective expansion of human NSC for transplantation.