Cellular and Molecular Neurobiology

, Volume 28, Issue 4, pp 511–517 | Cite as

Differential Centrifugation in Culture and Differentiation of Rat Neural Stem Cells

  • Sheng Ye
  • Zhi-peng Su
  • Jing Zhang
  • Xu Qian
  • Qi-chuan Zhuge
  • Yan-jun Zeng
Original Paper


(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.


Neural stem cell Differential centrifugation Single cell suspension 



We are grateful to the Nature Science Foundation of Zhejiang province (No. RC02062) for the continued support for our research on neural stem cells.


  1. Cavanagh JF, Mione MC, Pappas IS (1997) Basic fibroblast growth factor prolongs the proliferation of rat cortical progenitor cells in vitro without altering their cell cycle parameters. Cereb Cortex 7:293–302CrossRefPubMedGoogle Scholar
  2. Ciccolini F, Svendsen CN (1998) Fibroblast growth factor 2 (FGF-2) promotes acquisition of epidermal growth factor (EGF) responsiveness in mouse striatal precursor cells: identification of neural precursors responding to both EGF and FGF-2. J Neurosci 18:7869–7880PubMedGoogle Scholar
  3. Daadi M, Arcellana-Panlilio MY, Weiss S (1998) Activin co-operates with FGF2 to regulate tyrosine hydroxylase expression in the basal forebrain ventricular zone progenitors. Neurosci 86:867–880CrossRefGoogle Scholar
  4. Gritti A, Bonfanti L, Doetsch F, Caille I, Buylla AA, Lim DA, Galli R, Verdugo JMG, Herrera DG, Vescovi AL (2002) Multipotent neural stem cells reside into the rostral extension and olfactory bulb of adult rodents. J Neurosci 22:437–445PubMedGoogle Scholar
  5. Liu JM, Chen D, Meng XT (2004) A new method for isolation of neural stem cells. Chinese J Rehabil Theor Pract 10:25–26 + 72Google Scholar
  6. Luskin MB, Pearlman AL, Sanes JR (1988) Cell lineage in the cerebral cortex of the mouse studied in vivo and in vitro with a recombinant retrovirus. Neuron 1:635–647CrossRefPubMedGoogle Scholar
  7. Morrison SJ, Csete M, Groves AK, Melega W, Wold B, Anderson DJ (2000) Culture in reduced levels of oxygen promotes clonogenic sympathoadrenal differentiation by isolated neural crest stem cells. J Neurosci 20:7370–7376PubMedGoogle Scholar
  8. Pagano SF, Impagnatiello F, Girelli M, Cova L, Grioni E, Onofri M, Cavallaro M, Etteri S, Vitello F, Giombini S, Solero CL, Parati EA (2000) Isolation and characterization of neural stem cells from the adult human olfactory bulb. Stem Cells 18:295–300CrossRefPubMedGoogle Scholar
  9. Reynolds BA, Weiss S (1996) Colonal and populations analyses demonstrate that an EGF-responsive mammalian embryonic CNS precursor is a stem cell. Dev Biol 175:1–13CrossRefPubMedGoogle Scholar
  10. Sally T (2001) The development of neural stem cells. Nature 414:112–117CrossRefGoogle Scholar
  11. Satoh M, Sugino H, Yoshida T (2000) Activin promotes astrocytic differentiation of a multipotent neural stem cell line and an astrocyte progenitor cell line from murine central nervous system. Neurosci Lett 284:143–146CrossRefPubMedGoogle Scholar
  12. Shen Q, Zhong WM, Jan YN, Temple S (2002) Asymmetric numb distribution is critical for asymmetric cell division of mouse cerebral cortical stem cells and neuroblasts. Development 129:4843–4853PubMedGoogle Scholar
  13. Shih CC, Weng Y, Mamelak A, LeBon T, Hu MCT, Forman SJ (2001) Identification of a candidate human neurohematopoietic stem-cell population. Blood 98:2412–2422CrossRefPubMedGoogle Scholar
  14. Svendsen CN, ter Borg MG, Armstrong RJE, Rosser AE, Chandran S, Ostenfeld T, Caldwell MA (1998) A new method for the rapid and long term growth of human neural precursor cells. J Neurosci Methods 85:141–152CrossRefPubMedGoogle Scholar
  15. Tropepe V, Sibilia M, Ciruna BG, Rossant J, Wagner EF, Kooy DVD (1999) Distinct neural stem cells proliferate in response to EGF and FGF in the developing mouse. Dev Biol 208:166–188CrossRefPubMedGoogle Scholar
  16. Turner DL, Cepko CL (1987) A common progenitor for neurons and glia persists in rat retina late in development. Nature 328:131–136CrossRefPubMedGoogle Scholar
  17. Uchida N, Buck DW, He D, Reitsma MJ, Masek M, Phan TV, Tsukamoto AS, Gage FH, Weissman IL (2000) Direct isolation of human central nervous system stem cells. Proc Natl Acad Sci USA 97:14720–14725CrossRefPubMedGoogle Scholar
  18. Vescovi AL, Reynolds BA, Fraser DD, Weiss S (1993) bFGF regulates the proliferative fate of unipotent (neuronal) and bipotent (neuronal/astroglial) EGF-generated CNS progenitor cells. Neuron 11:951–966CrossRefPubMedGoogle Scholar
  19. Weiss S, Dunne C, Hewson J, Wohl C, Wheatley M, Peterson AC, Reynolds BA (1996) Multipotent CNS stem cells are present in the adult mammalian spinal cord and ventricular neuroaxis. J Neurosci 16:7599–7609PubMedGoogle Scholar
  20. Zheng ZH, Hu JS, Chen WL, Lin L, Zhong XR, Lin JY (2003) Ultrastructure of neural stem cell neurosphere cultured in vitro. Acta Anatomica Sinica 34:615–619Google Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  1. 1.Department of Neurosurgery, First Affiliated Hospital Wenzhou Medical CollegeWenzhouChina
  2. 2.Biomedical Engineering CenterBeijing University of TechnologyBeijingChina
  3. 3.Department of Histology and EmbryologyWenzhou Medical CollegeWenzhouChina

Personalised recommendations