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

Background: Neurobiology is studied by neuron cells from primary cultures or cell lines depending on the purpose of the research. Various methods were developed to obtain neuron cells in the cortex, hippocampus or from all brain tissue from the fetal brain or newborn mice. Neuron cells are unable to proliferate therefore the isolation of neuron progenitor cells (NPCs) needs to be developed. This study aims to develop a method of isolating NPCs from intact tissue of newborn mouse brains easily and practically. Methods: Brain tissue was obtained from Sprague Dawley rats aged 2 days. Experiments were carried out in two stages which included add trypsin 0,05% to brain tissue and then incubated for 10 minutes, adding culture medium, then filtered with pore size membrane and centrifuging for 10 minutes. The next step is to remove the supernatant then add with HBSS-glucose, put it in a 35% and 65% Ficoll solution and centrifugation, then the supernatant is planted in dish and then transferred again to the dish with poly-D-lysine cup. Characterization of neuron marker was carried out by immunocytochemistry (NeuN and microtubule-associated protein 2-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 more than 95% cells that obtained are expressing PSANCAM and A2B5. After 4 days culture, cells exhibit positive for neuron marker as MAP2 and NeuN. Conclusion: Successfully developed the easy and practical method to isolate NPCs from the whole brain of post-natal rat with high viability and purity. (Health Science Journal of Indonesia 2018;9(2):63-9) Keyword: post-natal rat, neuron progenitor cells (NPCs), isolation Health Science Journal of Indonesia Zainuri et al. 64 Neuron cells are needed for in vitro neurobiology studies such as neurotoxicology studies, molecular signaling pathway and pathogenesis studies of neurodegenerative. 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. The limited proliferation capacity of primary neurons necessitates further study to optimize neurons isolation techniques. In developing and adult mammalian brain, there are NPCs that maintain the regeneration process of neuronal cells. NPCs are multipotent cells committed to the neural lineage that can self-renew, proliferate and differentiate so it can be expanded in vitro. NPCs are normally quiescent and reside within specific niche including subventricular zone (SVZ) of the lateral ventricle, the subgranular zone of the dentate gyrus (DG) of the hippocampus and periventricular region surrounding the central canal of spinal cord. Various methods are performed to obtain NPCs from cortical, hippocampal and whole brain of adult, neonatal and prenatal rat. Each method has some effectiveness and advantages which is different. Isolation NPCs from specific region need high skill, the dissecting tool and stereomicroscope to cut SVZ and DG area of the one adult rat brain. This study aimed to develop an easy and practical protocol for NPCs isolation.