title: Optimal time for passaging neurospheres based on primary neural stem cell cultures.
abstract: Cultured neural stem cells (NSCs) provide a powerful means for investigating central nervous system disease, neuron development, differentiation, and regeneration. To obtain sufficient neurospheres, subculturing is essential following establishment of the primary NSC culture. Passaging the primary neurospheres is a key issue that is often ignored. We evaluated the influence of different passaging schedules on primary cultured NSCs. Passaging was performed on day 5, 7 or 9. We observed more neurospheres with diameters of 200-250 μm on day 7 than on day 5 or 9. Prolonging the time of primary culture reduced the cell metabolic activity by the MTT assay and cell proliferation by colony-forming assay and the differentiation to neurons from cells at P2 and later decreased. Additionally, more cells were in G0/G1 phase, and higher expression of p16                         (                            INK4a                         ) and lower expression of cyclin D1 was found when the time of primary culture was prolonged to 9 days compared to 7-days cultures. Thus, in this study, we established that the optimal time for subculturing aggregated NSCs was on day 7 based on the primary culture.
authors.affiliation: Department of Neurosurgery, Affiliated Hospital of Nantong University, 20 Xisi Road, Nantong, 226001, China.
authors.forename: Fangling
authors.initials: F
authors.lastname: Xiong
authors.forename: Huasong
authors.initials: H
authors.lastname: Gao
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhen
authors.forename: Xue
authors.initials: X
authors.lastname: Chen
authors.forename: Weiwei
authors.initials: W
authors.lastname: Lin
authors.forename: Jianhong
authors.initials: J
authors.lastname: Shen
authors.forename: Yaohua
authors.initials: Y
authors.lastname: Yan
authors.forename: Xiaodong
authors.initials: X
authors.lastname: Wang
authors.forename: Mei
authors.initials: M
authors.lastname: Liu
authors.forename: Yilu
authors.initials: Y
authors.lastname: Gao