Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

In accordance with New South Wales, Australia law, post mortem brain tissue was acquired from adult dogs euthanized for reasons unrelated to the study.

1. Preparation of Culture Medium

1. Prepare a 0.1% gelatin solution by adding 0.1 g of gelatin powder to 100 ml of distilled water and agitating at 37 °C until dissolved. Sterilize the solution by UV irradiation for 15 min. This media can then be stored at 4 °C for up to 1 month.
2. Prepare a 3:1 ratio of Dulbecco's Modified Eagle Medium (DMEM) and nutrient mixture F12 by adding 167 ml of F-12 to 500 ml of DMEM (4.5 g/L D-glucose). Add 6.7 ml of Penicillin/Streptomycin solution (for a 1% final concentration). This media can be stored in the dark at 4 °C for up to 1 month.
3. Prepare 500 ml of serum media by combining 440 ml of DMEM (4.5 g/L D-glucose), 5 ml of L-alanyl-L-glutamine dipeptide (200 mM), 5 ml of Penicillin/Streptomycin and 50 ml of fetal bovine serum (FBS). This media can be stored in the dark at 4 °C for up to 1 month.
4. Complete growth medium consists of Neural Stem Cell (NSC) Basal Medium and Proliferation Neural Supplement-A (NS-A), 2% FBS, 2 µg/ml heparin, 20 ng/ml epidermal growth factor (EGF) and 10 ng/ml basic fibroblast growth factor (bFGF). Make this media fresh, immediately prior to brain dissection, and warm to 37 °C in a water bath.

2. Brain Extraction

1. Begin extraction within 6 hr post mortem by sterilizing the dorsal surface of the dog's head using povidone-iodine.
2. Using a scalpel, make a longitudinal incision along the sagittal midline, across the entire dorsal surface of the cranium, to expose the underlying masseter muscles. The length of this cut will depend on the species and age of the dog.
3. At the rostral and caudal borders of the cranium, make additional 5 – 10 cm perpendicular cuts on both sides, passing behind the eyes and ears respectively, to allow the skin to be fully reflected.
4. Using a scalpel, separate the masseter muscles from their attachment to the sagittal crest of the skull and scrape any remaining connective tissue from the cranium.
5. Using an oscillating bone saw cut the cap of the skull in a circumferential line. Using a probe or scalpel sever any remaining attachments with the dura, and then carefully remove the skull cap to expose the dorsal surface of the brain.
   Caution: The thickness of the bone varies at different regions in the skull, so caution must be exercised to not penetrate too deep and damage the underlying brain.
6. Sever the spinal cord by inserting a scalpel between the upper cervical vertebrae.
7. With one hand to gently lift the brain, use a scalpel to carefully free the brain from the cranial fossae and sever the connecting nerves and blood vessels located on its ventral side. Gently lift the brain out of the skull and into a container of DPBS.
   Caution: The large olfactory bulbs of the canine brain may extend deep into the anterior cranial fossa. Care must be taken when removing the brain so as to preserve these structures.

3. Hippocampal Dissection

1. Rinse the brain in Dulbecco's phosphate buffered saline (DPBS) to remove any blood and place it dorsal side down onto a 150 mm Petri dish.
2. Slowly bisect the brain longitudinally through the mid-sagittal plane using a wet brain knife and a single slice that utilizes the full length of the blade. Do not apply additional downward pressure, or use a sawing motion, as this may damage the tissue.
3. Placing each hemisphere medial surface up, dissect out the hippocampus using a scalpel and fine forceps, and transfer it to a 35 mm tissue culture dish.

4. Isolation of Neural Precursor Cells

1. Mince the tissue samples using a scalpel blade into approximately 1 mm³ pieces.
2. Transfer the tissue into a 15 ml tube, add 2 ml of 0.1% Trypsin EDTA, and incubate for 7 min in a water bath at 37 °C.
3. Halt the enzyme reaction by adding 4 ml of serum media to the tube. Pellet the suspension by centrifugation at 100 x g for 7 min and remove the supernatant.
4. Resuspended the pellet in 300 µl of DPBS, and mechanically dissociated by gently pipetting up and down, using a 1,000 µl pipette tip, until a smooth homogenate forms.
5. Add 14 ml of serum media to the tube and pass the cell suspension through a 40 µm cell strainer. Pellet the suspension by centrifugation at 100 x g for 7 min, remove the supernatant, and resuspend in complete growth medium.

5. Neurosphere Culture

1. From the cell suspension obtained at isolation, count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.
2. Dilute the cell suspension in complete growth medium and seed at 1 x 10⁵ cells/cm² into uncoated wells of a 6 well plate.
3. Incubate at 37 °C, 5% CO₂ and > 95% humidity for 14 – 28 days, replace 50% of the growth media every 7 days. Measure the diameter of the neurospheres regularly. If allowed to grow beyond 100 µm in diameter, cell death and spontaneous differentiation can occur within the neurospheres.

6. Neurosphere Passage

1. To passage as a floating culture, when the neurospheres reach approximately 100 µm in diameter, combine all the neurosphere containing media into a single tube. Pellet by centrifugation at 100 x g for 7 min and remove the supernatant.
2. Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 °C. Gently pipette up and down 100 times, using a 200 µl pipette tip, to ensure dissociation of the neurospheres.
3. Halt the enzyme reaction by adding 5 ml of serum media to the tube and centrifuge at 100 x g for 7 min.
4. Remove the supernatant, resuspend the cell pellet in 1 ml complete growth medium and count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.
5. Dilute the cell suspension in complete growth medium and seed at 1 x 10⁵ cells/cm² into uncoated wells of a 6 well plate. Incubate at 37 °C, 5% CO₂ and >95% humidity for 14 – 28 days, replacing 50% of the growth media every 7 days.

7. Adherent Culture of Neural Precursor Cells

1. Add a sufficient volume of 0.1% gelatin solution to cover the surface of a tissue culture dish and cure in an incubator at 37 °C for 1 hr prior to processing the neurospheres for adherent culture.
2. From the floating neurosphere culture, combine all media into a single tube. Pellet the neurospheres by centrifugation at 100 x g for 7 min and remove the supernatant.
3. Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 °C. Gently pipette up and down 100 times, using a 200 µl pipette tip.
4. Add 5 ml of serum media to halt the enzyme reaction and centrifuge at 100 x g for 7 min. Discard the supernatant and resuspend the cell pellet in 1 ml complete growth medium and count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.
5. Dilute the cell suspension in complete growth medium, remove the gelatin from the tissue culture flasks and seed the cells at 1 x 10⁴ cells/cm².
6. Incubate the cells at 37 °C, 5% CO₂ and > 95% humidity. Replace the media with fresh, warmed, complete growth media every three days until the culture reaches approximately 80% confluence (after approximately 7 days).

8. Neural Colony Forming Assay

1. Combine Neural Colony Forming Cell (NCFC) Assay media components: NCFC serum free medium, Proliferation NS-A, and collagen solution (3 mg/ml). Supplement this media with 2 µg/ml heparin, 20 ng/ml EGF, and 10 ng/ml bFGF.
2. Following hippocampal tissue dissociation resuspend the cells in neural colony forming assay medium, pre-warmed to 37 °C in a water bath. Seed the cells at a clonal density of 1.1 x 10⁵ cells/ml into a 35 mm culture dish.
3. Incubate the cells in this semi-solid collagen matrix at 37 °C, 5% CO₂ and > 95% humidity. Culture for 28 days, replacing 50% of the growth media every 7 days.

9. Differentiation of Neural Precursor Cells

1. Add a sufficient volume of 5 µg/cm² laminin solution to cover the surface of a tissue culture dish and cure in an incubator at 37 °C for 24 hr before processing the cells for differentiation.
   Note: Remove the laminin solution and rinse the coated surface twice in DPBS immediately prior to seeding.
2. When passaging, seed the cell suspension at 1 x 10⁴ cells/cm² in DMEM-F12 (3:1) media (pre-warmed to 37 °C) supplemented with 20 ng/ml EGF and 40 ng/ml bFGF onto the laminin coated culture dish.
3. Incubate the cells at 37 °C, 5% CO₂ and > 95% humidity and allow cells to proliferate for 3 days.
4. To differentiate the adherent neural precursor cells, after 3 days replace the media with differentiation media containing DMEM-F12 (3:1) media pre-warmed to 37 °C, supplemented with 10 ng/ml brain derived neurotrophic factor (BDNF).
5. During differentiation replace 50% of the media every 3 days. Differentiation occurs gradually over approximately 21 – 28 days.
   Caution: Once the cells have begun to differentiate, only replace 50% of the media at one time as exposure to air can have a detrimental effect on the health of the cells.