Stem Cell Laboratory

Laboratory Protocols

Adherent culture of mouse subependymal zone

Isolation and culture of neural stem cells from the adult mouse SEZ

1. The animal is sacrificed by cervical dislocation, the brain is extracted and placed into in a 60-mm Petri dish with ice-cold dissection medium (10mM of HEPES in HBSS 1×).
2. With the ventral side facing up, the brain is coronally transected using a surgical blade at the level of the optic chiasm. 3. The ventricle from the caudal side is opened up using forceps, allowing to visualize the ventricular face of the wall of the lateral ventricle. The SEZ is located just beneath the surface formed by a single layer of ependymal cells.
3. The perimeter of the SEZ is cut and afterwards, a thin layer beneath the surface is dissected, avoiding as much as possible the adjacent striatal tissue. The SEZs from 3 mice are pulled together and placed in a 15-ml conical tube containing 10 ml of dissection medium.
4. The dissection medium is discarded by aspiration with a Pasteur pipette and replaced with 5 ml of filtered and pre-warmed (37 °C) dissociation medium (containing 0.7 mg/ml hyaluronic acid and 1.33 mg/ml trypsin HBSS with 2 mM glucose).
5. The samples are incubated without shaking at 37 °C for a total of 30 min. (After the first 15 minutes, a gentle trituration of the tissue into pieces is done by pipetting up and down with a 5-ml sterile disposable pipette).
6. The dissociation is stopped by adding an equal volume (~5ml) of solution 3 (consisting of 4% BSA in EBSS buffered with 20 mM HEPES)
7. For the removal of tissue debris, the cell suspension is passed through a 70-μm cell strainer. Centrifugation at 200g for 5 min.
8. The supernatant is discarded and re-suspended in 10 ml of ice-cold solution 2 (0.9 M sucrose in HBSS 1X). Centrifugation at 450g for 10 min.
9. The cells are re-suspended in 2 ml of ice-cold solution 3 (Note: A fresh 15-ml conical tube is filled with 12 ml of ice-cold solution 3 and the cell suspension is carefully transferred on the top of the new tube).
10. Cells are then centrifuged at 250g for 7 min. The supernatant is carefully removed and re-suspended in pre-warmed (37 °C) culture medium (DMEM/F12 Glutamax, supplemented with B27, 2 mM glutamine, 100 units/ml penicillin/streptomycin, buffered with 8 mM HEPES).
11. The cells are seeded onto poly-D-lysine–coated plastic coverslips. Note: Approximately 200–300 cells per mm2 (or the yield of one entire brain per well) are seeded on each coverslip.

 Protocol from Ortega et al., Nature Protocol, 2011

This work was supported by Aristeia II, GEMCCTR “Self-renewal and differentiation decisions in neural stem cells: Geminin, cell cycle control and transcriptional regulation”