Name: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors (Video) | JoVE Journal
Uploaded: 2016-11-29T15:00:00.000+00:00
Duration: 9 min 37 s
Description: Learn to isolate and expand adult neural precursor cells from the canine hippocampus for neurogenesis research. This method provides a valuable mammalian model with closer parallels to human brain function.

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5 minutes

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

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
<ol>
	<li>Prepare a 0.1% gelatin solution by adding 0.1 g of gelatin powder to 100 ml of distilled water and agitating at 37 &#176;C until dissolved. Sterilize the solution by UV irradiation for 15 min. This media can then be stored at 4 &#176;C for up to 1 month.</li>
	<li>Prepare a 3:1 ratio of Dulbecco&#39;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 &#176;C for up to 1 month.</li>
	<li>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 &#176;C for up to 1 month.</li>
	<li>Complete growth medium consists of Neural Stem Cell (NSC) Basal Medium and Proliferation Neural Supplement-A (NS-A), 2% FBS, 2 &#181;g/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 &#176;C in a water bath.</li>
</ol>
2. Brain Extraction
<ol>
	<li>Begin extraction within 6 hr post mortem by sterilizing the dorsal surface of the dog&#39;s head using povidone-iodine.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Sever the spinal cord by inserting a scalpel between the upper cervical vertebrae.</li>
	<li>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.</li>
</ol>
3. Hippocampal Dissection
<ol>
	<li>Rinse the brain in Dulbecco&#39;s phosphate buffered saline (DPBS) to remove any blood and place it dorsal side down onto a 150 mm Petri dish.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
4. Isolation of Neural Precursor Cells
<ol>
	<li>Mince the tissue samples using a scalpel blade into approximately 1 mm3 pieces.</li>
	<li>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 &#176;C.</li>
	<li>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.</li>
	<li>Resuspended the pellet in 300 &#181;l of DPBS, and mechanically dissociated by gently pipetting up and down, using a 1,000 &#181;l pipette tip, until a smooth homogenate forms.</li>
	<li>Add 14 ml of serum media to the tube and pass the cell suspension through a 40 &#181;m cell strainer. Pellet the suspension by centrifugation at 100 x g for 7 min, remove the supernatant, and resuspend in complete growth medium.</li>
</ol>
5. Neurosphere Culture
<ol>
	<li>From the cell suspension obtained at isolation, count the number of viable cells using Trypan blue dye exclusion and a hemocytometer.</li>
	<li>Dilute the cell suspension in complete growth medium and seed at 1 x 105 cells/cm2 into uncoated wells of a 6 well plate.</li>
	<li>Incubate at 37 &#176;C, 5% CO2 and &#62; 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 &#181;m in diameter, cell death and spontaneous differentiation can occur within the neurospheres.</li>
</ol>
6. Neurosphere Passage
<ol>
	<li>To passage as a floating culture, when the neurospheres reach approximately 100 &#181;m 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.</li>
	<li>Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 &#176;C. Gently pipette up and down 100 times, using a 200 &#181;l pipette tip, to ensure dissociation of the neurospheres.</li>
	<li>Halt the enzyme reaction by adding 5 ml of serum media to the tube and centrifuge at 100 x g for 7 min.</li>
	<li>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.</li>
	<li>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 &#176;C, 5% CO2 and &#62;95% humidity for 14 - 28 days, replacing 50% of the growth media every 7 days.</li>
</ol>
7. Adherent Culture of Neural Precursor Cells
<ol>
	<li>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 &#176;C for 1 hr prior to processing the neurospheres for adherent culture.</li>
	<li>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.</li>
	<li>Resuspend the pellet in 1 ml of 0.1% Trypsin EDTA and incubate for 7 min in a water bath at 37 &#176;C. Gently pipette up and down 100 times, using a 200 &#181;l pipette tip.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 and &#62; 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).</li>
</ol>
8. Neural Colony Forming Assay
<ol>
	<li>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 &#181;g/ml heparin, 20 ng/ml EGF, and 10 ng/ml bFGF.</li>
	<li>Following hippocampal tissue dissociation resuspend the cells in neural colony forming assay medium, pre-warmed to 37 &#176;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.</li>
	<li>Incubate the cells in this semi-solid collagen matrix at 37 &#176;C, 5% CO2 and &#62; 95% humidity. Culture for 28 days, replacing 50% of the growth media every 7 days.</li>
</ol>
9. Differentiation of Neural Precursor Cells
<ol>
	<li>Add a sufficient volume of 5 &#181;g/cm2 laminin solution to cover the surface of a tissue culture dish and cure in an incubator at 37 &#176;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.</li>
	<li>When passaging, seed the cell suspension at 1 x 104 cells/cm2 in DMEM-F12 (3:1) media (pre-warmed to 37 &#176;C) supplemented with 20 ng/ml EGF and 40 ng/ml bFGF onto the laminin coated culture dish.</li>
	<li>Incubate the cells at 37 &#176;C, 5% CO2 and &#62; 95% humidity and allow cells to proliferate for 3 days.</li>
	<li>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 &#176;C, supplemented with 10 ng/ml brain derived neurotrophic factor (BDNF).</li>
	<li>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.</li>
</ol>

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<li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells%5BTitle%5D)+AND+%22BMC+Dev+Biol%22%5BJournal%5D)">Cell-extracellular matrix interactions regulate neural differentiation of human embryonic stem cells.</a> BMC Dev Biol. 8 (90), 1-13 (2008).</li>
<li>Goffredo, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Setting+the+conditions+for+efficient%2C+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells%5BTitle%5D)+AND+%22Cell+Death+Differ%22%5BJournal%5D)">Setting the conditions for efficient, robust and reproducible generation of functionally active neurons from adult subventricular zone-derived neural stem cells.</a> Cell Death Differ. 15 (12), 1847-1856 (2008).</li>
</ol>

The authors have nothing to disclose.

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...

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.

<table><tbody><tr><td>1,000 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF1000</td><td></td></tr><tr><td>150 mm Petri dish</td><td>BD Biosciences</td><td>351058</td><td></td></tr><tr><td>15 ml centrifuge tubes</td><td>Greiner Bio One</td><td>188271</td><td></td></tr><tr><td>200 &amp;mu;l filtered pipette tip</td><td>Axygen</td><td>TF200</td><td></td></tr><tr><td>24 well culture plate</td><td>Greiner Bio One</td><td>662160</td><td></td></tr><tr><td>35 mm tissue culture dish</td><td>BD Biosciences</td><td>353001</td><td></td></tr><tr><td>40 &amp;micro;m cell strainer</td><td>BD Biosciences</td><td>352340</td><td></td></tr><tr><td>6 well culture plate</td><td>BD Biosciences</td><td>351146</td><td></td></tr><tr><td>B-27 Supplement (50x) serum free</td><td>Life Technologies&amp;nbsp;</td><td>17504044</td><td></td></tr><tr><td>Basic fibroblast growth factor (bFGF)</td><td>Life Technologies</td><td>13256029</td><td></td></tr><tr><td>Brain derived neurotrophic factor (BDNF)</td><td>Millipore</td><td>GF029</td><td></td></tr><tr><td>Collagen solution</td><td>Stem Cell Technologies</td><td>04902</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>DMEM (4.5 g/L, D-glucose) 500 ml</td><td>Life Technologies</td><td>11960044</td><td></td></tr><tr><td>DPBS</td><td>Life Technologies</td><td>14190250</td><td></td></tr><tr><td>Epidermal growth factor (EGF)</td><td>BD Biosciences</td><td>354001</td><td></td></tr><tr><td>F-12 nutrient mixture (Ham) (1x) Liquid</td><td>Life Technologies</td><td>31765035</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Life Technologies</td><td>16141079</td><td></td></tr><tr><td>Gelatin from Porcine Skin Type A</td><td>Sigma-Aldrich</td><td>G1890</td><td></td></tr><tr><td>L-alanyl-L-glutamine dipeptide (GlutaMAX)</td><td>Life Technologies</td><td>35050061</td><td></td></tr><tr><td>Heparin sodium salt from (porcine)</td><td>Sigma-Aldrich</td><td>H314950KU</td><td></td></tr><tr><td>Laminin (mouse)</td><td>Life Technologies</td><td>23017015</td><td></td></tr><tr><td>NCFC serum free medium (NeuroCult)</td><td>Stem Cell Technologies</td><td>5720</td><td>Also available in the Neurocult NCFC Assay Kit from Stem Cell Technologies. Cat: 05740&amp;nbsp;</td></tr><tr><td>Proliferation NS-A (NeuroCult)</td><td>Stem Cell Technologies</td><td>05773</td><td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&amp;nbsp; from Stem Cell Technologies.</td></tr><tr><td>NSC basal medium (Rat; NeuroCult)</td><td>Stem Cell Technologies</td><td>5770</td><td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&amp;nbsp;</td></tr><tr><td>Penicillin/Streptomycin (5,000 U/ml)</td><td>Life Technologies</td><td>15070063</td><td></td></tr><tr><td>Povidone-iodine</td><td>Munipharma</td><td>Betadine</td><td></td></tr><tr><td>Trypan blue (0.4%)</td><td>Life Technologies</td><td>15250061</td><td></td></tr><tr><td>Trypsin EDTA</td><td>Life Technologies</td><td>25200056</td><td></td></tr><tr><td>Class II biological safety cabinet</td><td>ThermoFisher Scientific</td><td>Safe 2020 1.2</td><td></td></tr><tr><td>Brain knife (disposable)</td><td>Macroknife</td><td></td><td></td></tr><tr><td>Cell culture incubator</td><td>ThermoFisher Scientific</td><td>HERAcell 150i</td><td></td></tr><tr><td>Centrifuge</td><td>Hettich</td><td>Universal 320R</td><td></td></tr><tr><td>Dumont #5 Forceps</td><td>Dumont</td><td></td><td></td></tr><tr><td>Easypet Electric pipette</td><td>Eppindorf&amp;nbsp;</td><td></td><td></td></tr><tr><td>Hemocytometer</td><td>Boeco</td><td>Bright-Line Improved Neubauer</td><td></td></tr><tr><td>Manual pipettes</td><td>Eppindorf research</td><td></td><td></td></tr><tr><td>Oscillating bone saw&amp;nbsp;</td><td></td><td></td><td></td></tr><tr><td>Scalpel blades (No.21)</td><td>Paramount</td><td></td><td></td></tr><tr><td>Scissors&amp;nbsp;</td><td>Delta</td><td></td><td></td></tr><tr><td>Water bath</td><td>Grant</td><td>JB Aqua 18 plus</td><td></td></tr></tbody></table>

isolation and expansion of adult canine hippocampal neural precursors

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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.

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...

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.

The rate of neurogenesis within the adult hippocampus has been shown to vary across mammalian species. The canine hippocampus, demonstrating a structural ...

The overall goal of this procedure is to effectively isolate and expand the population of neural precursor cells that reside in the adult canine hippocampus. This method can be used to obtain and study adult neural precursor cells, which then may help answer key questions in the field of adult neurogenesis. The main advantage of this technique is that it grants researchers access to an alternative higher mammal animal model, which may have closer parallels to the human brain than traditional animal models.In this procedure, begin extraction within six hours post mortem by sterilizing the dorsal surface of the animals head using povidone iodine. 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. At the rostral and caudal borders of the cranium make additional five to 10 centimeter perpendicular cuts on both sides, passing behind the eyes and ears respectively to allow the skin to be fully reflected.Separate the masseter muscles from their attachment to the sagittal crest of the skull and scrape any remaining tissue from the cranium. Using an osculating bone saw, cut the cap of the skull in a circumferential line. Than using a probe or a scalpel, sever any remaining attachments with the dera and carefully remove the skull cap to expose the dorsal surface of the brain.After that, sever the spinal cord by inserting a scalpel between the upper cervical vertebrae. Gently lift the brain and use a scalpel to carefully free the brain from the cranial fossa. Sever the connective nerves and blood vessels located on it's ventral side.Then, gently transfer the brain to a container of DPBS. To dissect the hippocampus, rinse the brain in DPBS to remove any blood. Next, place it dorsal side down onto a 150 millimeter Petri dish.Slowly bisect the brain longitudinally through the midsagittal plane using a wedge brain knife cutting with 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. Place both hemispheres medial surface up.Dissect at the hippocampi using a scalpel and fine forceps and transfer them to a 35 millimeter tissue culture dish. Mince the tissue samples into approximately one cubic millimeter pieces using a scalpel blade. Next, transfer the tissue into a 15 milliliter tube.Add two millimeters of 0.1%trypsin EDTA and incubate it for seven minutes in a water bath at 37 degrees Celsius. Afterward, halt the enzyme reaction by adding four milliliters of serumedia to the tube. Pellet the suspension by centrifugation at 100 times g for seven minutes and then remove the supernatant.Re-suspend the pellet in 300 microliters of DPBS and mechanically dissociate it by gently pipetting up and down until a smooth homogenate has formed. Subsequently add 14 milliliters of serumedia to the tube and pass the cell suspension through a 40 micron cell strainer. Next, pellet the suspension by centrifugation at 100 times g for seven minutes.Remove the supernatant and re-suspend it in complete growth medium. From the cell suspension obtained at isolation, count the number of viable cells using trypan blue dye exclusion and a hemacytometer. Dilute the cell suspension in complete growth medium and seat the cells in the uncoated wells of a six well plate.Incubate the cells at 37 degrees Celsius for 14 to 28 days and replace 50%of the growth media every seven days. Measure the diameter of the neuro spheres regularly and allow the cells to grow to no more than 100 micrometers in diameter. To passage as a floating culture, when the neuro spheres reach approximately 100 micrometers in diameter, combine all the neuro sphere containing media into a single tube.Pellet the cells by centrifugation at 100 times g for seven minutes and remove the supernatant. Next, re-suspend the pellet in one milliliter of 0.1%trypsin EDTA and incubate it for seven minutes in the water bath at 37 degrees Celsius. Afterward, gently pipet up and down 100 times using a 200 microliter pipet tip to ensure dissociation of the neuro spheres.Halt the enzyme reaction by adding five milliliters of serumedia to the tube and centrifuge it at 100 times g for seven minutes. After seven minutes remove the supernatant, re-suspend the cell pellet in 1 milliliter of complete growth medium and count the number of viable cells. Subsequently, dilute the cell suspension in complete growth medium and seat the cells in the uncoated wells of a six well plate.Incubate the cells at 37 degrees Celsius for 14 to 28 days and replace 50%of the growth medium every seven days. In this procedure, add a sufficient amount of 0.1%gelatin solution to cover the tissue culture dish surface and cure it in an incubator at 37 degrees Celsius for one hour prior to processing the neuro spheres for adherent culture. Next, combine all the media from the floating neuro sphere culture into a single tube.Pellet the neuro spheres by centrifugation at 100 times g for seven minutes and then remove the supernatant. Re-suspend the pellet in one milliliter of 0.1%trypsin EDTA and incubate it for seven minutes in a water bath at 37 degrees Celsius. Gently pipet up and down 100 times using a 200 microliter pipet tip.After that, add five milliliters of seramedia to halt the enzyme reaction and centrifuge it at 100 times g for seven minutes. Discard the supernatant and re-suspend the cell pellet in 1 milliliter of complete growth medium. Then count the number of viable cells using trypan blue dye exclusion and a hemacytometer.Next, dilute the cell suspension in complete growth medium remove the gelatin from the tissue culture flasks and seed the cells. Afterward, incubate the cells at 37 degrees Celsius. Replace the media with fresh, warmed complete growth media every three days until the culture reaches approximately 80%confluence after approximately seven days.Neuro precursor cells were able to proliferate as floating neuro spheres or when seeded at one times tenth to the fourth cells per square centimeter onto 0.1%gelatin coated culture flasks as an adherent monolayer. No morphological differences were observed between dorsal and ventral hippocampal neuro precursor cells and both adherent cultures were able to undergo under 10 population doublings without any observed slow down in passage time. Nestin and Sox2 neural stem cell gene expressions in adherent culture were not significantly different across the hippocampal dorsal axis.When cultured in BDNF for 28 days, adult canine neuro precursors from both the dorsal and ventral hippocampus differentiate into mature neuronal encyclical cell types. While attempting this procedure, it's important to remember work quickly and maintain sterile working conditions. After watching this video, you should have a good understanding of how to isolate and expand neuro precursor cells from the adult canine hippocampus.

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Developmental Biology

Neural stem cells were prepared from the hippocampus of adult non-hibernating yearling Arctic ground squirrels (AGS). These neural stem cells can be expanded through numerous passages, differentiated and maintained as a nearly 50:50 neuron to glial culture.

Growth and Differentiation of Adult Hippocampal Arctic Ground Squirrel Neural Stem Cells

This video protocol demonstrates the neurosphere assay method to generate and expand neural stem cells from the adult mouse periventricular region, and provides technical insights to ensure one can achieve reproducible neurosphere cultures.

Isolation and Expansion of the Adult Mouse Neural Stem Cells Using the Neurosphere Assay

This video demonstrates the process of neurosphere formation and their expansion from hippocampal tissue. Neural stem and progenitor cells from the postnatal mouse dentate gyrus tissue proliferate and form primary neurospheres, which are then reseeded to form secondary neurospheres.

Formation and Expansion of Neurospheres from a Hippocampal Tissue

Source: Gonzalez-Ramos, A., et. al., Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders. J. Vis. Exp. (2022)This video demonstrates the stereotaxic injection of human-derived interneuron precursor cells into the hippocampus of a genetically modified mouse pup with reduced inhibitory interneurons, aiming to restore excitatory-inhibitory balance.

Transplantation of Human-Derived Interneuron Precursor Cells into the Mouse Pup Hippocampus

During adult hippocampal neurogenesis, a distinct set of genetic markers are expressed when a quiescent neural stem cell sequentially progresses and develops into a functionally integrated neuron in the circuit. Using heat-induced antigen retrieval, progenitor cell types that are otherwise difficult to detect are identified with improved effectiveness.

Heat-Induced Antigen Retrieval: An Effective Method to Detect and Identify Progenitor Cell Types during Adult Hippocampal Neurogenesis

Here we describe a detailed protocol for the simultaneous generation of neural precursor cell cultures, as either adherent monolayers or neurospheres, from the subventricular zone and dentate gyrus of individual adult mice.

One Mouse, Two Cultures: Isolation and Culture of Adult Neural Stem Cells from the Two Neurogenic Zones of Individual Mice

This video article illustrates a comprehensive protocol to detect and quantify all stages of adult hippocampal neurogenesis within the same tissue section. We elaborated a method to overcome the limitations of indirect multiple immunofluorescence that arise when suitable antibodies from different host species are unavailable.

Immunohistochemistry and Multiple Labeling with Antibodies from the Same Host Species to Study Adult Hippocampal Neurogenesis

Establishing culture systems for the expansion of adult neural stem cells (aNSCs) allows for the examination and application of aNSCs for therapy. The subcallosal zone (SCZ) has recently been recognized as a novel neuroblast-forming region in adult mice. Here, methods for the isolation, expansion, and differentiation of SCZ-aNSCs are described.

Isolation and Culture of Adult Neural Stem Cells from the Mouse Subcallosal Zone

The goal of this protocol is to describe an approach for analyzing behavior of adult neural stem/progenitor cells in response to chemogenetic manipulation of a specific local neural circuit.

Assaying Circuit Specific Regulation of Adult Hippocampal Neural Precursor Cells

In this article, we describe, in detail, a protocol for the generation of neurosphere cultures from postnatal mouse neural stem cells derived from the main mouse neurogenic niches. Neurospheres are used to identify neural stem cells from brain tissue allowing the estimation of precursor cell numbers. Moreover, these 3D structures can be plated in differentiative conditions, giving rise to neurons, oligodendrocytes and astrocytes, allowing the study of cell fate.

Isolation and Expansion of Neurospheres from Postnatal (P1&#8722;3) Mouse Neurogenic Niches

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival rate.

Isolation, Expansion, and Nucleofection of Neural Stem Cells from Adult Murine Subventricular Zone

Learn to isolate and expand adult neural precursor cells from the canine hippocampus for neurogenesis research. This method provides a valuable mammalian model with closer parallels to human brain function.

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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