A new method to culture primary neural stem cells of embryonic rats

Chengming XING; Xiuli GENG; Yuxia LIU

ABSTRACT

BACKGROUND:

Both in vivo and vitro microenvironment can influence the proliferation and differentiation of neural stem cells (NSCs). In addition, cell culture solution plays variable roles in cell proliferation and differentiation.

OBJECTIVE:

To develop a convenient and rapid method to promote NSC primary culture by modifying traditional serum-free medium. DESIGN, TIME AND

SETTING:

Randomized controlled cell trial was performed at Institute of Brain Science, Qingdao University Medical School from November 2005 to September 2006.MATERIALSTen Wistar rat of gestation for 12-16 days; cell suspension fron brain tissue of embryonic rat.

METHODS:

Cell suspension were seeded into four 50-mL culture flasks with cell density of 1×106 mL-1, and divided into 2 groups with 2 flasks in each group. The control cells (flasks A and B) were cultured in serum-free medium containing DMEM/F12, B27 (2%), basic fibroblast growth factor (20 μg/L), and epidermal growth factor (20μg/L), and the experimental cells (flasks C and D) were firstly cultured with DMEM/F12 containing fetal bovine serum (FBS, 5%), following by the same serum-free medium 2 to 3 days later. MAIN OUTCOME

MEASURES:

The proliferation of NSCs was observed by inverted microscopy and immunocytochemistry.

RESULTS:

①Most of the cells in two culture conditions expressed nestin, the specific marker of NSCs, and were immunocytochemically positive. ②Neural stem cells cultured with FBS formed neurospheres 3-4 days earlier than those without FBS. ③There were no significant differences in cell number, but the neurospheres under the experimental condition were larger than those in control group. Some of the cells were neurone specific enolase-positive after serum-conditioned induction, and some were glial fibrillary acidic protein-positive, indicating NSCs had differentiated into neuron-like cells and neurogliocytes.

CONCLUSION:

FBS-conditioned pre-culture can accelerate the proliferation of neural stem cells.