Extended serial passaging of mammalian neural stem cells in suspension bioreactors

Neural stem cells (NSCs) are primitive cells that are the 'parent' cells of all the cells in the central nervous system (CNS). Their discovery in 1992 opened the door to a multitude of potential therapies and treatments to cure neurodegenerative diseases such as Parkinson's disease, multiple sclerosis, and Huntington's disease, which affect millions of people worldwide and cost billions of dollars in health care each year. This study proposes optimal serial passaging protocols so that mammalian neural stem cells can effectively be grown in suspension culture. We examined stationary culture passaging protocols and developed our own optimal procedure. Also examined was the effect of serially cultivating the neural stem cells in suspension culture for an extended period of time. The cells were grown for over 35 days in suspension with an overall multiplication ratio of over 10⁷ with no decrease in growth rate, maximum cell density, or viability. The cells also remained karyotypically normal through 25 doublings and retained their ability to be differentiated into all the major cell types of the CNS - neurons, astrocytes, and oligodendrocytes. For the first time, mammalian neural stem cells were grown on a larger scale in suspension culture and maintained their stem cell characteristics. A semicontinuous scheme for large-scale production is also presented.