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Method Article

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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DOI:

10.3791/54953

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November 29th, 2016

 ,  ,  , 
1Regenerative Neuroscience Group, University of Sydney, 2Wellcome Trust Centre for Neuroimaging, Institute of Neurology, University College London

In This Article

Summary

The canine brain is a valuable model in which to study adult neurogenesis. Presented here are protocols for isolating and expanding adult canine hippocampal neural precursor cells from primary brain tissue.

Abstract

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural intermediacy between the rodent and human hippocampi, is therefore a valuable model in which to study adult neurogenesis. In vitro culture assays are an essential component of characterizing neurogenesis and adult neural precursor cells, allowing for precise control over the cellular environment. To date however, culture protocols for canine cells remain under-represented in the literature. Detailed here are systematic protocols for the isolation and culture of hippocampal neural precursor cells from the adult canine brain. We demonstrate the expansion of canine neural precursor cells as floating neurospheres and as an adherent monolayer culture, producing stable cell lines that are able to differentiation into mature neural cell types in vitro. Adult canine neural precursors are an underused resource that may provide a more faithful analogue for the study of human neural precursors and the cellular mechanisms of adult neurogenesis.

Introduction

Regional variations in the rate of neurogenesis have been observed along the dorsoventral axis of the rodent hippocampus1,2. Furthermore, the rates of hippocampal neurogenesis also show distinct inter-species variation, with precursor cell turnover in the subgranular zone shown to be significantly lower in adult humans than in rodents3-5. Inter-species differences in hippocampal structural anatomy may be relevant here, as it has been postulated that neural stem cell distribution along the murine ventricular neuraxis may be influenced by cephalic flexures during embryological development6. To date, the rodent brain remains the most popular system in which to study adult neurogenesis. However, the brain of the domestic dog (Canis familiaris), with a size and structural organization intermediate between that of humans and rodents7, represents a valuable yet highly underused animal model. The canine hippocampus in particular embodies this structurally intermediate nature8-10 and can provide a unique perspective on intrinsic variations in neural precursor cell populations. With many closer parallels to the human brain, the canine model may also offer insight into the biology of adult human neurogenesis.

In vitro culture assays have become a key tool for the study of neural precursors and the cellular and biomolecular processes of adult neurogenesis. The neurosphere assay and adherent monolayer culture represent the two predominant systems for expanding neural precursor cells in vitro11-13. Protocols for brain extraction, hippocampal microdissection or neural precursor culture assays have been well documented for the rodent model14-16. However, for the adult canine brain they remain comparatively few17,18, focused instead on fetal or neonatal tissue19-21.

In our published study7 we investigated regional variations in neurogenesis and neural precursor cell populations across the dorsoventral axis of the adult canine hippocampus. Although highly dependent on breed, adulthood in canines is reached between 1 and 3 years of age. Here, we present detailed methods for the extraction, isolation and culture of neural precursor cells from the canine hippocampus. We provide systematic protocols for the expansion of neural precursor cells as both floating neurospheres and as an adherent monolayer culture, and for their subsequent differentiation into mature neural cell types.

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Protocol

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.

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Results

Through the use of in vitro neural precursor assays, neurogenesis and neural precursor cell populations were characterized and compared across the dorsoventral axis of the adult canine hippocampus. Neural precursor cells derived from isolated hippocampal tissue formed floating neurospheres within 14 days of isolation, reaching a diameter of 100 µm by 28 days of culture. Neurospheres derived from dorsal and ventral isolates showed no difference in mean size, and following enzymati...

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Discussion

The protocols described here are optimized to maintain favorable culture conditions for maximizing cell viability. The speed and care taken during extraction, isolation and expansion is of critical importance. A critical step for establishing adherent monolayer expansion is the effective dissociation of the primary neurospheres. Following passage, insufficiently dissociated neurospheres may generate secondary floating neurospheres. During media change, these neurospheres may be removed, dissociated and reseeded for adher...

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Disclosures

The authors have nothing to disclose.

Acknowledgements

This work was supported by the National Health and Medical Research Council (NHMRC) of Australia grants (#568969 and 1004152).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
1,000 μl filtered pipette tipAxygenTF1000
150 mm Petri dishBD Biosciences351058
15 ml centrifuge tubesGreiner Bio One188271
200 μl filtered pipette tipAxygenTF200
24 well culture plateGreiner Bio One662160
35 mm tissue culture dishBD Biosciences353001
40 µm cell strainerBD Biosciences352340
6 well culture plateBD Biosciences351146
B-27 Supplement (50x) serum freeLife Technologies 17504044
Basic fibroblast growth factor (bFGF)Life Technologies13256029
Brain derived neurotrophic factor (BDNF)MilliporeGF029
Collagen solutionStem Cell Technologies04902Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740 
DMEM (4.5 g/L, D-glucose) 500 mlLife Technologies11960044
DPBSLife Technologies14190250
Epidermal growth factor (EGF)BD Biosciences354001
F-12 nutrient mixture (Ham) (1x) LiquidLife Technologies31765035
Fetal bovine serum (FBS)Life Technologies16141079
Gelatin from Porcine Skin Type ASigma-AldrichG1890
L-alanyl-L-glutamine dipeptide (GlutaMAX)Life Technologies35050061
Heparin sodium salt from (porcine)Sigma-AldrichH314950KU
Laminin (mouse)Life Technologies23017015
NCFC serum free medium (NeuroCult)Stem Cell Technologies5720Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740 
Proliferation NS-A (NeuroCult)Stem Cell Technologies05773Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771  from Stem Cell Technologies.
NSC basal medium (Rat; NeuroCult)Stem Cell Technologies5770Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771 
Penicillin/Streptomycin (5,000 U/ml)Life Technologies15070063
Povidone-iodineMunipharmaBetadine
Trypan blue (0.4%)Life Technologies15250061
Trypsin EDTALife Technologies25200056
Class II biological safety cabinetThermoFisher ScientificSafe 2020 1.2
Brain knife (disposable)Macroknife
Cell culture incubatorThermoFisher ScientificHERAcell 150i
CentrifugeHettichUniversal 320R
Dumont #5 ForcepsDumont
Easypet Electric pipetteEppindorf 
HemocytometerBoecoBright-Line Improved Neubauer
Manual pipettesEppindorf research
Oscillating bone saw 
Scalpel blades (No.21)Paramount
Scissors Delta
Water bathGrantJB Aqua 18 plus

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