Differential centrifugation in culture and differentiation of rat neural stem cells

(1) The study of neural stem cells (NSC) has attracted much attention in recent years because of their therapeutic potential. However, the problem in culture and differentiation of NSC was how to obtain single cell suspension that preserves the function of NSC, and remove the debris caused by mechanical dissociation. In the present study, we try to find a simple and effective way to address the problem, i.e. differential centrifugation. (2) After a gentle mechanical dissociation using Pasteur pipette, the suspension was first centrifuged at 100 g for 5 min, and then recentrifuged at 400 g for 6 min. Finally, the two deposits were resuspended and seeded into culture flask respectively. The suspension from the second deposit was allowed for further culture and differentiation. Immunofluorescence technique was used to identify neural stem cell, neuron, astrocyte, and oligodendrocyte. (3) After the second differential centrifugation, single cell suspension was obtained with 2-3 cell clusters, and the cells not only grew to form neurospheres, but also differentiated into neurons, astrocytes, and oligodendrocytes. (4) Differential centrifugation is a simple and effective way to obtain single cell suspension, which will help make large-scale production of neurodifferentiated cells more effective.