Optimization of primary culture of neural stem cells

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Author:
LAN Yan-xian(Department of Pharmacology, Nanjing General Hospital of Nanjing Military Command, Southern Medical University, Nanjing 210002, China)
LI Jin-heng(Department of Pharmacology, Nanjing General Hospital of Nanjing Military Command, Southern Medical University, Nanjing 210002, China)
WANG Ke-wan()
WANG Yong()
Journal Title:
CHINESE JOURNAL OF NEUROMEDICINE
Issue:
Volume 10, Issue 05, 2011
DOI:
10.3760/cma.j.issn.1671-8925.2011.05.007
Key Word:
Neural stem cell;Hippocampus;Trypsinization;Proliferation;Apoptosis

Abstract: Objective To study the influence of digestion times of low concentration trypsin on the proliferation and apoptosis of neural stem cells (NSCs) in the hippocampus of neonate rats.Methods Hippocampus of neonatal rats (within 24 h) were taken out, and treated with trypsin at 1.25g/L concentration and 37 ℃ for 5, 10, 15, 20 and 25 minutes; unicellular suspension was then successfully got and primary culture and subculture were performed. Effects of trypsinization on cell viability and growth of NSCs were compared by observing the cell morphology and Trypan blue staining.The 5-bromodeoxyuridine labeling was performed to assess the self-renewing and proliferative activities of NSCs. Fluorescence immunocytochemistry was carried out to examine the expressions of BrdU and nestin. Apoptosis was measured by Annexin V-FITC/PI assay and flow cytometry. Results Primary and passage culture of NSCs enjoyed rapid proliferation and formation of neurospheres. The neurosphere cells expressed NSCs specific marker nestin by immunofluorescence; all the neurosphere cells could incorporate BrdU into the nucleus; of the neurospheres obtained from the 3rd, 5th and 7th d, those digested for 15 rain enjoyed the highest level of NSCs neurospheres, the highest BrdU labeled clone and the lowest cell apoptosis as compared with those digested for 5, 10, 20 and 25 min (P<0.05). Conclusion The NSCs isolated from the hippocampus of neonatal rats have the ability of proliferation in vitro. And 1.25 g/L concentration of trypsin with digestion times could positively change the proliferative and apoptosis capacity of NSCs: too short or long digestion times can inhibit the proliferation of NSCs and induce the apoptosis of NSCs; the longer the digestion time, the higher the apoptosis of NSCs.

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