An easy and cost-effective method for the isolation and culturing of neural stem/progenitor cells from the subventricular (SVZ) and dentate gyrus (DG) of adult mouse brain.

ABSTRACT

BACKGROUND: Isolation of adult Neural Stem/Progenitor Cells (NSPCs) from their neurogenic niches, is a prerequisite for studies involving culturing of NSPCs as neurospheres or attached monolayers in vitro. The currently available protocols involve the use of multiple animals and expensive reagents to establish the NSPCs culture. NEW METHOD: This unit describes a method to isolate and culture NSPCs from the two neurogenic niches in the mouse brain, the Subventricular Zone (SVZ) and Dentate gyrus (DG)/subgranular zone (SGZ), in an easy and cost-effective manner. RESULTS: NSPCs from SVZ and DG regions of adult mouse brains were isolated and cultured up to passage 15 without losing their stem/progenitor characteristics. These NSPCs could be differentiated into neurons, astrocytes, and oligodendrocytes, revealing its trilineage potential. COMPARISON WITH EXISTING METHODS: This protocol eliminates the need for multiple animals as well as the use of many expensive reagents mentioned in previous protocols, adding to the cost-effectiveness of experiments. In addition, we have effectively reduced the number of steps involved in isolation and propagation, thereby minimizing the chances of contamination. CONCLUSION: Our simplified protocol for the isolation and culturing of adult NSPCs from the SVZ and DG demonstrates a cost-effective and efficient alternative to existing methods, reducing the need for sacrificing many animals and the usage of expensive reagents. This method permits the long-term maintenance of NSPCs' stem/progenitor characteristics and their effective differentiation into the major types of cells in the brain, making it a valuable resource for researchers in the field. BASIC PROTOCOL Isolation and Culturing of Neural Stem/Progenitor cells from the Sub ventricular Zone and the Dentate Gyrus of the adult mouse brain. SUPPORT PROTOCOL 1 Cryopreservation, and revival of frozen NSPCs. SUPPORT PROTOCOL 2 Preparation of adherent monolayer cultures of neural stem/progenitor cells for the differentiation into multiple lineages SUPPORT PROTOCOL 3 Differentiation of NSPCs to neuronal and glial lineages SUPPORT PROTOCOL 4 Characterization of differentiated cells by immunocytochemistry.