Optimal time for passaging neurospheres based on primary neural stem cell cultures
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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.
KeywordsNeurosphere Neural stem cells Primary culture Cell passage Optimal time
This study was supported by a grant from the National Natural Science Foundation of China (No. 81070992), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Foundation of the Graduate Natural Science Innovation Project of Nantong University (No. YKC10050).
Conflict of interest
This study was supported by a grant from the National Natural Science Foundation of China (No. 81070992), the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD) and the Foundation of the Graduate Natural Science Innovation Project of Nantong University (No. YKC10050). The authors have declared that no conflict of interest exists.
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