Simple Method to Isolation and Culture of Neuron Progenitor Cells (NPCs) from Whole Brain Post-Natal Rat

Abstract

Background: Using of neuron cells for in vitro neurobiology study is needed. Neuron cell can be obtained from a primary neuron or neuronal cell lines, depend on the aim of the study because both are not equivalent. Various methods are performed to obtain primary neurons from the cortical, hippocampal and whole brain of pre or neonatal rat. The limitations of neuron cells to proliferate so that is necessary to develop a method to isolate neuron progenitor cells (NPCs). The aim of the present study was to isolate NPCs from whole brain post-natal rat.   Methods: Whole brain were obtained from neonates Sprague Dawley rat. There are 2 step to get NSC; first isolation by taking the brain into the 15 ml of tube with 1 ml of  0,05% trypsin EDTA for 400g brain (incubated in the 370C, 5% CO2 for 10 minutes),  tirturation with adding 1 ml culture medium  and 5 ml HBSS-glucose then filtered by 70μm pore size membrane and centrifuged  2000 rpm for 10 minutes. Second: remove of supernatant with add 1 ml of HBSS-Glucose and taking it into a tube with  35% and 65% concentration of Ficoll then centrifuged at 1800 g for 10 minutes then supernatant were replated twice with poly D lysine (100µg/ml). Characterization of progenitor neuron immunotype was checked by immunohistochemistry with positive marker (NeuN and MAP2) and flow cytometry (PSANCAM+ and A2B5 -). Results: In this study, our result show that this method does not take longer than one hours and > 95% cells that obtained are expressing PSANCAM+.  After 4 days culture, cells exhibit positive for neuron marker as MAP2 and NeuN.   Conclusion: The method that our develope to isolate neuron progenitor cell from whole-brain are more effective and more simple with high viability and purity.