Formation and Expansion of Neurospheres from a Hippocampal Tissue

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

1. Basic setup and preparation of culture medium

1. On the day of dissection, prepare the appropriate amount of growth medium corresponding to serum-free medium (SFM) composed of Dulbecco's modified eagle medium [(DMEM)/F12 with L-glutamine] (Table of Materials) supplemented with 100 U/mL penicillin and 100 µg/mL streptomycin (pen/strep), 1% B27, with also 10 ng/mL epidermal growth factor (EGF) and 5 ng/mL basic fibroblast growth factor (bFGF). Warm the culture medium to 37 °C in a water bath.    
   NOTE: The volume of growth medium depends on the number of pups, for 5 pups prepare ~100 mL (50 mL for subventricular zone (SVZ) and 50 mL for dentate gyrus (DG)); however, after counting the number of cells (step 4.1) the exact volume will have to be adjusted.
2. For microdissection of SVZ and DG, prepare the calcium and magnesium-free Hanks' balanced saline solution (HBSS) dissection medium supplemented with 100 U/mL penicillin/streptomycin (pen/strep).       
   NOTE: Prepare 50−100 mL of dissection medium.
3. Set up a dissection microscope and prepare the tools needed to remove the brain (scissors and small spatula) and for SVZ and DG microdissections (Dumont small scissors, #7 forceps, #5 forceps, #5S forceps) by soaking in 70% ethanol.

2. Harvesting of postnatal (P1−3) mouse brains and SVZ/DG microdissections

1. Prepare 60 mm Petri dishes (growth area 21 cm²) with HBSS supplemented with pen/strep and 2 sample tubes (one for SVZ and one for DG) with 500 µL of supplemented HBSS each.
2. Euthanize mice pups (P1−3) according to the protocol approved by the Institutional Animal Care facility/guidelines. Perform decapitation with a single incision with sharp scissors at the base of the brainstem.
3. Holding the ventral part of the body at the base of the head and using small, pointed scissors, make a midline incision in the skin over the entire length of the head, thus revealing the surface of the skull.
4. Make a longitudinal incision at the base of the skull and continue cutting along the sagittal suture using small scissors with an angle shallow as possible in order to avoid damaging the brain structures.
5. Peel the skull to the sides using curved forceps and expose the brain.
   CAUTION: Make sure the dissecting instruments are free of ethanol before touching the brain.
6. Isolate the brain from the skull using a small spatula, by sliding under the base of the brain to cut the cranial nerves and blood vessels that are connected to the base of the brain, and transfer the brain into a Petri dish containing cold supplemented HBSS solution.
7. Place the Petri dish containing the brain under a dissecting microscope at low magnification and position the brain on its dorsal surface.
8. Using fine forceps, remove the meninges from the ventral side of the brain and the olfactory bulbs, while holding the brain in position by the cerebellum. Rotate the brain onto the ventral aspect and peel off the rest of the meninges.
   NOTE: Removing the dorsal meninges is a crucial step to ensure correct brain slicing.
9. Discard the cerebellum making a cut using forceps. Place a filter paper with a pore size of 11 µm onto a tissue chopper (Table of Materials) and set the brain onto the filter paper using curved-pointed forceps. Chop the brain into 450 µm coronal sections and use a wet lamina to collect the sectioned brain into a new Petri dish filled with cold supplemented HBSS.
10. To dissect out the SVZ, use forceps to separate coronal slices in an anterior-to-posterior fashion until reaching slices with the lateral ventricles, under a dissecting microscope.
11. Cut the thin layer of tissue surrounding the lateral wall of the ventricles (which corresponds to the SVZ) with fine forceps, excluding the striatal parenchyma and the corpus callosum. Isolate the SVZ by placing the tip of the forceps in the lateral corners of the lateral ventricle: one immediately under the corpus callosum and the other into the tissue immediately adjacent to the ventral area of the lateral ventricle. Then, cut a small line of tissue surrounding the lateral ventricle.
12. Collect the dissected tissue into a sample tube with supplemented HBSS solution previously identified as SVZ.
    NOTE: Exclude the SVZ in slices where both the lateral ventricles and the hippocampal formation begin to appear.
13. Go through all slices after SVZ microdissection in an anterior-to-posterior fashion and reach the hippocampal formation. Using forceps discard the first slice with hippocampus where DG is still unrecognizable.
14. To remove the DG, first isolate the hippocampus from the slices. Then, refocus the microscope to visualize the borders around the DG.
15. Dissect the DG portion by performing a cut between the DG and CA1 region followed by a vertical cut between the DG and CA3 region using forceps. Remove fimbria and any adjacent tissue.  
    NOTE: In P1−3 animals, the DG is almost undistinguishable from Ammon's horn but displays a small tip.
16. Collect the dissected tissue into a sample tube containing supplemented HBSS solutions previously identified as DG.       
    NOTE: Overall injury to the hippocampus or surrounding area will make it more difficult to isolate the DG. Using an atlas of the postnatal mouse brain is essential when the user is not familiar with isolating the SVZ and DG tissue from coronal sections.

3. Tissue dissociation

1. To dissociate the SVZ and DG tissue present in their respective tubes, add trypsin-ethylenediamine tetraacetic acid (trypsin-EDTA) 0.05% to have a final concentration of 5−10% of Trypsin-EDTA 0.05% in HBSS. Incubate for approximately 15 min at 37 °C, until the tissue is clumped together.
2. Wash the tissue from the trypsin by removing the media and adding 1 mL of new HBSS-supplemented solution for 4 consecutive times.
3. Remove the HBSS and resuspend the digested tissue in 1 mL of SFM supplemented with 10 ng/mL EGF and 5 ng/mL bFGF. Mechanically dissociate the pellet by gently pipetting up and down approximately 7−10x using a P1000 pipette, until getting a homogenous cell solution.    
   CAUTION: Excessive mechanical dissociation can lead to increased cell death and will negatively impact subsequent cell growth.

4. Expansion of postnatal neural stem cells as neurospheres

1. To determine the density of the dissociated SVZ or DG cell suspension (obtained in section 3), count the cells using a hematocytometer.
2. Dilute SVZ and DG single cell suspension at a density of 2 x 10⁴ cells/mL in SFM supplemented with 10 ng/mL EGF and 5 ng/mL bFGF. Seed SVZ and DG cells in uncoated 60 mm Petri dishes with a final volume of 5 mL/Petri dish.
3. Incubate SVZ and DG cells for 6−8 days and 10−12 days, respectively to form primary neurospheres, at 37 °C with 5% CO₂.    
   NOTE: Incubation days more than those mentioned can promote aggregation of neurospheres and higher levels of cell death in the center of the neurosphere.
4. When the majority of neurospheres have a diameter of 150−200 µm, perform the neurosphere passage.   
   NOTE: Passaging neurospheres when they do not have an appropriate diameter compromises all the next steps.

5. Passaging of neurospheres

NOTE: The following protocol can be applied to expand both SVZ and DG neurospheres.

1. To passage neurospheres, collect the SFM with growth factors containing neurospheres from the 60 mm Petri dish(es) and centrifuge for 5 min at 300 x g.
2. Discard the supernatant and resuspend the neurosphere pellet using a chemical dissociation kit (mouse) according to the manufacturer's instructions (Table of Materials).
   NOTE: Observe the incubation times precisely as they are crucial for performance.
3. Centrifuge for 5 min at 300 x g, remove the supernatant and add 1 mL of SFM supplemented with 10 ng/mL EGF and 5 ng/mL bFGF.
4. Triturate up and down approximately 10x with a P1000 pipette to dissociate neurospheres.
5. Count the number of cells using a solution containing 0.2% Trypan blue and a hematocytometer.
6. Reseed cells at a density of 2 x 10⁴ cells/mL in uncoated 60 mm Petri dishes.
7. Incubate SVZ and DG cells for 6−8 days and 10−12 days, respectively to obtain secondary neurospheres, at 37 °C with 5% CO₂.       
   NOTE: The self-renewal capacity of SVZ- and DG-derived NSPCs can be accessed by following protocol sections 5 and 6. For that, seed SVZ and DG cells at a density of 1.0 x 10⁴ cells/mL (in uncoated 24-well plates) in growth SFM medium containing 5 ng/mL EGF and 2.5 ng/mL bFGF (low EGF/bFGF). Count the number of resulting primary and secondary neurospheres.