Name: isolation and expansion of adult canine hippocampal neural precursors
Uploaded: 2016-11-29T15:00:00
Description: Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

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

I enlighet med New South Wales, Australien lag, post mortem hjärnvävnad förvärvades från vuxna hundar avlivades något samband med studien. 1. Beredning av odlingsmedium <ol><li> Bered en 0,1% gelatinlösning genom att tillsätta 0,1 g gelatinpulver till 100 ml destillerat vatten och omrörning vid 37 ° C tills det är upplöst. Sterilisera lösningen genom UV-bestrålning under 15 minuter. Detta media kan sedan lagras vid 4 ° C i upp till en månad. </li><li> Bered en 3: 1 förhållande av Dul...

<ol>
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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurogenesis+in+the+adult+human+hippocampus.">Neurogenesis in the adult human hippocampus.</a> Nat Med. 4 (11), 1313-1317 (1998).</li><li>Bull, N. D., Bartlett, P. 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A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+neurons+and+astrocytes+from+isolated+cells+of+the+adult+mammalian+central+nervous+system.">Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system.</a> Science. 255 (5052), 1707-1710 (1992).</li><li>Ray, J., Raymon, H. K., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+culturing+of+precursor+cells+and+neuroblasts+from+embryonic+and+adult+central+nervous+system.">Generation and culturing of precursor cells and neuroblasts from embryonic and adult central nervous system.</a> Methods Enzymol. 254, 20-37 (1995).</li><li>Oliver-Dela Cruz, J., Ayuso-Sacido, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cells+from+mammalian+brain:+isolation+protocols+and+maintenance+conditions.">Neural stem cells from mammalian brain: isolation protocols and maintenance conditions.</a> INTECH Open Access Publisher. , (2012).</li><li>Pacey, L., Stead, S., Gleave, J., Tomczyk, K., Doering, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+stem+cell+culture:+neurosphere+generation,+microscopical+analysis+and+cryopreservation.">Neural stem cell culture: neurosphere generation, microscopical analysis and cryopreservation.</a> Nat Protoc. , (2006).</li><li>Azari, H., Rahman, M., Sharififar, S., Reynolds, B. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+expansion+of+the+adult+mouse+neural+stem+cells+using+the+neurosphere+assay.">Isolation and expansion of the adult mouse neural stem cells using the neurosphere assay.</a> J Vis Exp. (45), e2393 (2010).</li><li>Walker, T. L., Kempermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One+mouse,+two+cultures:+isolation+and+culture+of+adult+neural+stem+cells+from+the+two+neurogenic+zones+of+individual+mice.">One mouse, two cultures: isolation and culture of adult neural stem cells from the two neurogenic zones of individual mice.</a> J Vis Exp. (84), e51225 (2014).</li><li>Lim, J. -. H., Koh, S., Olby, N. J., Piedrahita, J., Mariani, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+neural+progenitor+cells+from+adult+canine+brains.">Isolation and characterization of neural progenitor cells from adult canine brains.</a> Am J Vet Res. 73 (12), 1963-1968 (2012).</li><li>Herranz, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spontaneously+Arising+Canine+Glioma+as+a+Potential+Model+for+Human+Glioma.">Spontaneously Arising Canine Glioma as a Potential Model for Human Glioma.</a> J Comp Pathol. 154 (2), 169-179 (2016).</li><li>Walton, R. M., Parmentier, T., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neural+precursor+cell+regions+in+the+rostral+subventricular+zone,+hippocampal+subgranular+zone+and+cerebellum+of+the+dog+(Canis+lupus+familiaris).">Postnatal neural precursor cell regions in the rostral subventricular zone, hippocampal subgranular zone and cerebellum of the dog (Canis lupus familiaris).</a> Histochem Cell Biol. 139 (3), 415-429 (2013).</li><li>Walton, R. M., Wolfe, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Abnormalities+in+neural+progenitor+cells+in+a+dog+model+of+lysosomal+storage+disease.">Abnormalities in neural progenitor cells in a dog model of lysosomal storage disease.</a> J Neuropathol Exp Neurol. 66 (8), 760-769 (2007).</li><li>Milward, E. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+transplantation+of+multipotential+populations+of+epidermal+growth+factor-responsive,+neural+progenitor+cells+from+the+canine+brain.">Isolation and transplantation of multipotential populations of epidermal growth factor-responsive, neural progenitor cells from the canine brain.</a> J Neurosci Res. 50 (5), 862-871 (1997).</li><li>Ray, J., Peterson, D. A., Schinstine, M., Gage, F. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proliferation,+differentiation,+and+long-term+culture+of+primary+hippocampal+neurons.">Proliferation, differentiation, and long-term culture of primary hippocampal neurons.</a> Proc. Natl. Acad. Sci. 90 (8), 3602-3606 (1993).</li><li>Palmer, T., Ray, J., Gage, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF-2-responsive+neuronal+progenitors+reside+in+proliferative+and+quiescent+regions+of+the+adult+rodent+brain.">FGF-2-responsive neuronal progenitors reside in proliferative and quiescent regions of the adult rodent brain.</a> Mol Cell Neurosci. 6 (5), 474-486 (1995).</li><li>Zhu, G., Mehler, M., Mabie, P., Kessler, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+changes+in+progenitor+cell+responsiveness+to+cytokines.">Developmental changes in progenitor cell responsiveness to cytokines.</a> J. Neurosci. Res. 56 (2), 131-145 (1999).</li><li>Whittemore, S. R., Morassutti, D. J., Walters, W. M., Liu, R. -. H., Magnuson, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitogen+and+substrate+differentially+affect+the+lineage+restriction+of+adult+rat+subventricular+zone+neural+precursor+cell+populations.">Mitogen and substrate differentially affect the lineage restriction of adult rat subventricular zone neural precursor cell populations.</a> Exp. Cell. Res. 252 (1), 75-95 (1999).</li><li>Kennedy, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postmortem+survival+characteristics+of+rat+glial+cells+in+culture.">Postmortem survival characteristics of rat glial cells in culture.</a> J Neurol Neurosurg Psychiatry. 50 (6), 798-800 (1987).</li><li>Viel, J. J., McManus, D. Q., Cady, C., Evans, M. S., Brewer, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Temperature+and+time+interval+for+culture+of+postmortem+neurons+from+adult+rat+cortex.">Temperature and time interval for culture of postmortem neurons from adult rat cortex.</a> J. Neurosci. Res. 64 (4), 311-321 (2001).</li><li>Huat, T. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+enhances+cell+proliferation+and+survival+during+early+differentiation+of+mesenchymal+stem+cells+to+neural+progenitor-like+cells.">IGF-1 enhances cell proliferation and survival during early differentiation of mesenchymal stem cells to neural progenitor-like cells.</a> BMC Neurosci. 15 (91), 1-13 (2014).</li><li>Supeno, N. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=IGF-1+acts+as+controlling+switch+for+long-term+proliferation+and+maintenance+of+EGF/FGF-responsive+striatal+neural+stem+cells.">IGF-1 acts as controlling switch for long-term proliferation and maintenance of EGF/FGF-responsive striatal neural stem cells.</a> Int. J. Med. Sci. 10 (5), 522-531 (2013).</li><li>Ahmed, S., Reynolds, B. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BDNF+enhances+the+differentiation+but+not+the+survival+of+CNS+stem+cell-derived+neuronal+precursors.">BDNF enhances the differentiation but not the survival of CNS stem cell-derived neuronal precursors.</a> J Neurosci. 15 (8), 5765-5778 (1995).</li><li>Wohl, C. A., Weiss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Retinoic+acid+enhances+neuronal+proliferation+and+astroglial+differentiation+in+cultures+of+CNS+stem+cell-derived+precursors.">Retinoic acid enhances neuronal proliferation and astroglial differentiation in cultures of CNS stem cell-derived precursors.</a> J Neurobiol. 37 (2), 281-290 (1998).</li><li>Araujo, D. M., Cotman, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Trophic+effects+of+interleukin-4,-7+and-8+on+hippocampal+neuronal+cultures:+potential+involvement+of+glial-derived+factors.">Trophic effects of interleukin-4,-7 and-8 on hippocampal neuronal cultures: potential involvement of glial-derived factors.</a> Brain Res. 600 (1), 49-55 (1993).</li><li>Jensen, J. B., Parmar, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strengths+and+limitations+of+the+neurosphere+culture+system.">Strengths and limitations of the neurosphere culture system.</a> Mol Neurobiol. 34 (3), 153-161 (2006).</li><li>Singec, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defining+the+actual+sensitivity+and+specificity+of+the+neurosphere+assay+in+stem+cell+biology.">Defining the actual sensitivity and specificity of the neurosphere assay in stem cell biology.</a> Nat Methods. 3 (10), 801-806 (2006).</li><li>Conti, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Niche-independent+symmetrical+self-renewal+of+a+mammalian+tissue+stem+cell.">Niche-independent symmetrical self-renewal of a mammalian tissue stem cell.</a> PLoS Biol. 3 (9), 283 (2005).</li><li>Pollard, S. M., Conti, L., Sun, Y., Goffredo, D., Smith, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Adherent+neural+stem+(NS)+cells+from+fetal+and+adult+forebrain.">Adherent neural stem (NS) cells from fetal and adult forebrain.</a> Cereb Cortex. 16, 112-120 (2006).</li><li>Ma, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-extracellular+matrix+interactions+regulate+neural+differentiation+of+human+embryonic+stem+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Setting+the+conditions+for+efficient,+robust+and+reproducible+generation+of+functionally+active+neurons+from+adult+subventricular+zone-derived+neural+stem+cells.">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>

De protokoll som beskrivs här är optimerade för att bibehålla goda odlingsbetingelser för att maximera cellernas livskraft. Hastigheten och omsorg under extraktion, isolering och expansion är av kritisk betydelse. Ett kritiskt steg för att fastställa vidhäftande monolager expansion är en effektiv dissociation av de primära neuro. Efter passage, otillräckligt dissocierade neurosfärer kan generera sekundära flytande neurosfärer. Under media förändring, kan dessa neuro tas bort, dissocieras och såddes fö...

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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			<td height="21" style="height:21px;width:337px;">1000 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF1000</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">150 mm petri dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL centrifuge tubes</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">200 &#956;L filtered pipette tip</td>
			<td style="width:196px;">Axygen</td>
			<td style="width:252px;">TF200</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well culture plate</td>
			<td style="width:196px;">Greiner Bio One</td>
			<td style="width:252px;">662160</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">35 mm tissue culture dish</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">353001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">352340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6 well culture plate</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351146</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">17504044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Basic fibroblast growth factor (bFGF)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">13256029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMEM (4.5g/L, D-glucose) 500mL</td>
			<td style="width:196px;">Life Technologies&#160;</td>
			<td style="width:252px;">11960044&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Epidermal growth factor (EGF)</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">354001</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">F-12 nutrient mixture (Ham) (1X) Liquid</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">31765035</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum (FBS)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">16141079</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gelatin from Porcine Skin Type A</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">G1890</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">L-alanyl-L-glutamine dipeptide (GlutaMAX)</td>
			<td>Life Technologies</td>
			<td style="width:252px;">35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Heparin sodium salt from (porcine)</td>
			<td style="width:196px;">Sigma-Aldrich</td>
			<td style="width:252px;">H314950KU</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Laminin (mouse)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">23017015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Proliferation NS-A (NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">05773</td>
			<td>Also available in the Neurocult NCFC Assay Kit Cat: 05740, and NS-A Prolieration Kit (Rat) Cat: 05771&#160; from Stem Cell Technologies.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NSC basal medium (Rat; NeuroCult)</td>
			<td>Stem Cell Technologies</td>
			<td style="width:252px;">5770</td>
			<td>Also available in the Neurocult NS-A Prolieration Kit (Rat) from Stem Cell Technologies. Cat: 05771&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Penicillin/Streptomycin (5000 U/mL)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15070063</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Povidone-iodine</td>
			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Trypan blue (0.4%)</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">15250061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin EDTA</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">25200056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Class II biological safety cabinet</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">Safe 2020 1.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Brain knife (disposable)</td>
			<td style="width:196px;">Macroknife</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Cell culture incubator</td>
			<td style="width:196px;">ThermoFisher Scientific</td>
			<td style="width:252px;">HERAcell 150i</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
			<td style="width:196px;">Hettich</td>
			<td style="width:252px;">Universal 320R</td>
			<td style="width:915px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dumont #5 Forceps</td>
			<td style="width:196px;">Dumont</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Easypet Electric pipette</td>
			<td style="width:196px;">Eppindorf&#160;</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hemocytometer</td>
			<td style="width:196px;">Boeco</td>
			<td style="width:252px;">Bright-Line Improved Neubauer</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
			<td style="width:196px;">Eppindorf research</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Oscillating bone saw&#160;</td>
			<td style="width:196px;"></td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Scalpel blades (No.21)</td>
			<td style="width:196px;">Paramount</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:337px;">Scissors&#160;</td>
			<td style="width:196px;">Delta</td>
			<td style="width:252px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Water bath</td>
			<td style="width:196px;">Grant</td>
			<td style="width:252px;">JB Aqua 18 plus</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Genom användning av in vitro-neurala prekursorceller analyser, neurogenes och neurala prekursorceller cellpopulationer karakteriserades och jämfördes över dorsoventral axeln av den vuxna canine hippocampus. Neurala prekursorceller härledda från isolerade hippocampus vävnad bildade flytande neurosfärer inom 14 dagar efter isolering och nådde en diameter av 100 um med 28 dagars odling. Neurosfärer erhållna från dorsala och ventrala isolat visade ingen skillnad i medels...

Watch this Scientific Journal Video about Isolation and Expansion of Adult Canine Hippocampal Neural Precursors at JoVE.com

Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

Den hund hjärnan är en värdefull modell för att studera vuxen neurogenes. Presenteras här är protokoll för att isolera och expandera vuxna hund hippocampus neurala prekursorceller från primär hjärnvävnad.

Isolering och utbyggnad av vuxen hund Hippocampus neurala prekursorer

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

Research

JoVE Journal

Developmental Biology

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Ex Utero Elektroporation och Organotypic Slice Kultur av Mouse hippocampus Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Isolering av neurala stamceller / progenitorceller från periventrikulär regionen vuxen råtta och humant ryggmärgs

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of ...

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Stabil och effektiv genetisk modifiering av celler i vuxen mus V-SVZ för analys av neurala stamceller autonoma och icke-självständiga effekter

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex Vivo Culture of Chick Cerebellar Slices and Spatially Targeted Electroporation of Granule Cell Precursors

Ex vivo Kultur av Chick Cerebellar skivor och rumsligt riktade elektroporering av Granulat cellprekursorer

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying ...

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

Isolering och kultur av vuxna Zebrafish Brain-härledda Neuro

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Culturing Human Pluripotent and Neural Stem Cells in an Enclosed Cell Culture System for Basic and Preclinical Research

Odling av humana pluripotenta och neurala stamceller i en sluten cellodlingssystem för Basic och preklinisk forskning

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety ...

Isolation and Expansion of Mesenchymal Stem/Stromal Cells Derived from Human Placenta Tissue

Isolering och Expansion av mesenkymala stam / stromaceller celler från Human Placenta Tissue

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose ...

Stimulation of Notch Signaling in Mouse Osteoclast Precursors

Stimulering av Notch signalering i mus osteoklastprekursorer

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro ...

The Isolation and Culture of Primary Epicardial Cells Derived from Human Adult and Fetal Heart Specimens

Isolering och kultur av primära epikardiell celler från mänskliga vuxen och fostrets hjärta exemplar

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of ...

Derivation and Differentiation of Canine Ovarian Mesenchymal Stem Cells

Härledning och differentiering av hundarnas cystor mesenkymala stamceller

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies ...