Isolation Protocol of Mouse Neural Stem Cells

• Neural stem cells (NSCS) are special cells with self-renewal and multidirectional differentiation potential.
• It not only can be used to explore the molecular mechanism of nervous system development, but also can be used as an alternative means for the treatment of central nervous system injury, degenerative diseases, brain tumors and other diseases to establish a stable and reliable neural stem cell culture model in vitro, which is an effective means to explore the mechanism of proliferation, differentiation and in vivo transplantation of neural stem cells.

• Subependymal zone (SEZ) fragments are transferred from each brain into 15 mL sterile tubes with wide-perforated sterile plastic pipettes and wait for them to settle to the bottom of the tubes before the DPBS are removed.
• Each brain is added 1 mL of filtered papain solution and incubated in a 30℃ bath for 37 minutes to allow tissue digestion.
• 3 mL of control medium is added to dilute papain and stop digestion.
• Centrifuge at 100g for 2 minutes and carefully remove the supernatant by using a sterile plastic pipette. If a suction pump is used in this step, be careful to prevent suction of tissue.
• 3 mL of control culture medium is added and gently dissociated by using a fire-polished glass Pasteur pipette until the cell suspension is uniform. Alternatively, 1 mL of control medium is added and carefully dissociated through the p1000 micropipette suction. In both cases, keep the pipette at a constant rate to avoid bubbles.
• Use control culture medium to reach an 8-10 mL volume. Mix 1-2 times by slowly inverting the tube. Don't vortex.
• Centrifuge at 200g for 10 minutes. There should be a dense precipitate at this point, although the supernatant may be a little cloudy due to cell debris.
• Suck out as much supernatant as possible without disturbing the precipitation.
• The individual cells are re-suspended by adding 1mL of preheated complete culture medium and carefully pipetting up and down through the p1000 micropipette suction until the precipitates are completely depolymerized. Avoid bubbles forming.
• For the standard primary culture, cells obtained from two SEZs are assigned to 48 wells of the P8-well plate (the growth area is 1 cm² per well). 3 mL of complete culture medium is added to the tube, and 500 μL of cell suspension is mixed and plated into each well. If possible, avoid inoculating cells in peripheral pores and fill these empty pores with sterile water or DPBS to avoid evaporation of the medium.
• Incubate in 2 humidified incubators for 7-10 days. During this time, the differentiated cells will die, while the NSCs and some progenitor cells will begin to proliferate and form neuro-spheres.

• Yield can be estimated five days after inoculation by manually counting primary neuro-spheres on an inverted microscope with a phase-out optical element. Each brain will have between 2,000 and 4,000 neuro-spheres, depending on anatomical accuracy.
• All steps must be performed in a laminar flow cabinet under strict aseptic conditions.

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