Download Neonatal rat organotypic brain slice culture and its effect on induced

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Organotypic slice culture of neonatal rat cortex and induced
neural stem cell differentiation
*
Qian Jiao, Hai-xia Zhang, Hai-Xia Lu , Yong Liu
*
Institute of Neurobiology, Xi'an Jiaotong University College of Medicine, Xi'an
710061, China
* Corresponding author
E-mail: [email protected], [email protected]
Abstract:
Objective During neural development, neural stem cell (NSC) undergoes a precise
proliferation and differentiation process. This process is directed by various
factors from the NSC niche. In order to investigate the effect of spatial signal
on NSC differentiation, in the present study, the survival and differentiation of
CM-DiI labeled NSCs were observed on the organotypic slice cultures from neonatal
rat cortex.
Methods SD rats on 3 to 5 postnatal days were sacrificed according to the ethical
guidelines of the NIH Regulations for Experimentation on Laboratory Animals and set
out by the Xi’an Jiaotong University. Brain slices with 200μm thickness were cut
on a vibrotome microtome and cultured in modified DMEM/F12 serum free medium at 37℃
with 95% O2 and 5% CO2. Organotypic slice cultures were observed under inverted
phase-contrast microscope every day and the medium was half refreshed every 3 to
4 days. NSCs were isolated from the cortex of rat embryos on embryonic day 14.5 and
cultured in DMEM/F12 serum free medium supplemented with B27 and N2. NSCs on passage
number 3 were labeled by CM-DiI for 20 minutes in dark and cultured for 3 to 4 days.
The labeled cells were transplanted onto the organotypic slices in 2 weeks culture.
The survival of transplanted NSCs was confirmed by visual CM-DiI labeled cells on
the slices. The differentiation of transplanted NSCs was identified by
immunofluorescence staining and the DiI/GFAP or DiI/β tubullin III double labeled
cells were considered to be astrocytes or neurons.
Results The organotypic slice cultures survived well for at least 4 weeks in the
modified medium and became clear vision on 10 days later, which possibly due to the
elimination of microglia. CM-DiI labeled NSCs were observed on the slice cultures
4 days after transplantation. These labeled NSCs differentiated into GFAP+ glia and
β tubullin III+ neurons.
Conclusion Our results suggest that the organotypic slice culture might produce some
specific signals to induce the transplanted NSCs to differentiate into a certain
cell type. The further study about NSCs differentiation on organotypic slice
cultures from different brain regions will reveal the effect of niche in NSC
behavior.
Keywords: organotypic brain slice; neural stem cells; rat; differentiation