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

Gemäß New South Wales, Australien Gesetz, post mortem Hirngewebe wurde von erwachsenen Hunden aus Gründen in keinem Zusammenhang mit der Studie euthanasiert erworben. 1. Herstellung von Kulturmedium <ol><li> Bereiten einer 0,1% igen Gelatinelösung durch Zugabe von 0,1 g Gelatinepulver zu 100 ml destilliertem Wasser und Rühren bei 37 ° C bis gelöst. Sterilisieren der Lösung durch UV-Bestrahlung für 15 min. Diese Medien können dann für bis zu 1 Monat bei 4 ° C gelagert werden. </li><li> Vorbereiten eines 3: 1-Verhältnis von Dulbeccos modifiziertem Eagle-Medium (DMEM) und Nährstoffgemisch F12 durch Zugabe von 167 ml F-12 bis 500 ml DMEM (4,5 g / L D-glucose). Hinzufügen 6,7 ml Penicillin / Streptomycin-Lösung (für eine Endkonzentration von 1%). Dieses Medium kann für bis zu 1 Monat bei 4 ° C im Dunkeln gelagert werden. </li><li> Bereiten 500 ml Serum Medien durch die Kombination 440 ml DMEM (4,5 g / L D-glucose), 5 ml L-Alanyl-L-Glutamin-Dipeptid (200 mM) 5 ml Penicillin / Streptomycin und 50 ml fötales Bovine Serum (FBS). Dieses Medium kann für bis zu 1 Monat bei 4 ° C im Dunkeln gelagert werden. </li><li> Komplettes Wachstumsmedium besteht aus Neural Stem Cell (NSC) Basal Medium und Proliferation Neural Supplement-A (NS-A), 2% FBS, 2 ug / ml Heparin, 20 ng / ml epidermaler Wachstumsfaktor (EGF) und 10 ng / ml basischer Fibroblasten-Wachstumsfaktor (bFGF). Machen Sie diese Medien frisch, unmittelbar vor der Gehirnsezierung, und warm bis 37 ° C in einem Wasserbad. </li></ol> 2. Gehirn Extraction <ol><li> Beginnen Extraktion innerhalb von 6 Stunden post mortem durch die dorsale Oberfläche des Kopfes des Hundes Sterilisieren mit PVP-Jod. </li><li> Mit einem Skalpell machen einen Längsschnitt entlang der sagittalen Mittellinie über die gesamte Rückenfläche des Schädels, die zugrunde liegenden Kaumuskeln zu belichten. Die Länge dieser Schnitt wird von der Art und Alter des Hundes ab. </li><li> An den rostralen und kaudalen Grenzen des Schädels, machen zusätzliche 5 - 10 cm senkrecht Schnitte auf beiden Seiten, übergebening hinter den Augen und Ohren sind, zu ermöglichen, die Haut vollständig reflektiert werden. </li><li> Mit einem Skalpell, trennen Sie die Kaumuskeln aus ihrer Befestigung an der Sagittalkamm des Schädels und kratzen alle verbleibenden Bindegewebe aus dem Schädel. </li><li> ein oszillierendes Knochen Mit sah die Kappe des Schädels in einer Umfangslinie geschnitten. Mit Hilfe einer Sonde oder einem Skalpell durchtrennen alle verbleibenden Anlagen mit der Dura, und dann vorsichtig die Schädelkappe entfernen Sie die dorsale Oberfläche des Gehirns aufzudecken. Achtung: Die Dicke des Knochens variiert in den verschiedenen Regionen in den Schädel, so dringen Vorsicht geboten ausgeübt werden , um nicht zu tief und das darunter liegende Gehirn schädigen. </li><li> Sever das Rückenmark durch ein Skalpell zwischen der oberen Halswirbel stecken. </li><li> Mit der einen Hand, um das Gehirn sanft heben, verwenden Sie ein Skalpell vorsichtig auf das Gehirn aus dem Schädelgrube befreien und durchtrennen die Verbindungs ​​Nerven und Blutgefäße befindet sich an seiner Bauchseite. Heben Sie die bregen aus dem Schädel und in einen Behälter mit DPBS. Achtung: Die großen Riechkolben des Hunde-Gehirn tief in die vordere Schädelgrube erstrecken kann. Es muss darauf geachtet werden, wenn das Gehirn zu entfernen, um diese Strukturen zu erhalten. </li></ol> 3. Hippocampale Dissection <ol><li> Spülen Sie das Gehirn in Dulbeccos phosphatgepufferter Salzlösung (DPBS) kein Blut zu entfernen und sie Rückenseite nach unten auf eine 150 mm Petrischale. </li><li> bisect langsam das Gehirn längs durch die Mitte Sagittalebene einen nassen Gehirn Messer und eine einzelne Scheibe verwenden, die über die gesamte Länge der Klinge verwendet. Wenden Sie keine zusätzlichen Abwärtsdruck, oder eine Sägebewegung, da dies das Gewebe beschädigen können. </li><li> Platzieren jeder Hemisphäre medial Fläche nach oben, sezieren aus dem Hippocampus eines Skalpells und einer feinen Pinzette verwenden und übertragen sie auf eine 35-mm-Gewebekulturschale. </li></ol> 4. Isolierung von neuralen Vorläuferzellen <ol><li> Mince die tAusgabe Proben mit einem Skalpell in ca. 1 mm 3 Stück verwenden. </li><li> Übertragen Sie das Gewebe in einen 15-ml-Röhrchen, 2 ml 0,1% Trypsin-EDTA, und Inkubation für 7 Minuten in einem Wasserbad bei 37 ° C. </li><li> Halt die Enzymreaktion durch Zugabe von 4 ml von Serum Medien auf das Rohr. Pellet die Suspension durch Zentrifugation bei 100 g für 7 min und entfernen Sie den Überstand. </li><li> Resuspendierte das Pellet in 300 ul DPBS und mechanisch durch vorsichtiges Pipettieren bis distanzierte und unten, eine 1000 ul Pipettenspitze, bis eine glatte Homogenat Formen. </li><li> In 14 ml Serum Medien in das Röhrchen und übergeben Sie die Zellsuspension durch ein Sieb 40 &amp; mgr; m-Zelle. Pellet die Suspension durch Zentrifugation bei 100 xg für 7 min, um den Überstand zu entfernen, und resuspendieren in komplettem Wachstumsmedium. </li></ol> 5. Neurosphäre Kultur <ol><li> Von der Zellsuspension bei Isolierung erhalten, zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss verwenden und eine HämoZytometer. </li><li> Verdünne die Zellsuspension in komplettem Wachstumsmedium und Samen bei 1 x 10 5 Zellen / cm2 in unbeschichteten Vertiefungen einer Platte mit 6 Vertiefungen. </li><li> Inkubieren bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit für 14 - alle 7 Tage 28 Tage 50% der Wachstumsmedien ersetzt. Messen Sie den Durchmesser der regelmäßig Neurospheres. Wenn erlaubt über 100 &amp; mgr; m im Durchmesser zu wachsen, Zelltod und spontane Differenzierung in den Neurosphären auftreten. </li></ol> 6. Neurosphäre Passage <ol><li> Um Passage als schwimmender Kultur, wenn die Neurosphären etwa 100 &amp; mgr; m im Durchmesser erreichen, kombinieren alle Neurosphere haltigen Medien in einem einzigen Rohr. Pellet durch Zentrifugation bei 100 g für 7 min und entfernen Sie den Überstand. </li><li> Resuspendieren des Pellets in 1 ml 0,1% Trypsin EDTA und Inkubation bei 37 ° C für 7 min in einem Wasserbad. Pipette vorsichtig nach oben und unten 100-mal, eine 200 ul Pipettenspitze, um sicherzustellen, dissociatiauf der Neurosphären. </li><li> Halt die Enzymreaktion durch Zugabe von 5 ml Serum Medien in das Röhrchen und zentrifugiert bei 100 g für 7 min. </li><li> Entfernen Sie den Überstand, Zellpellet in 1 ml komplettem Wachstumsmedium und zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss Verwendung und einer Zählkammer. </li><li> Verdünne die Zellsuspension in komplettem Wachstumsmedium und Samen bei 1 x 10 5 Zellen / cm2 in unbeschichteten Vertiefungen einer Platte mit 6 Vertiefungen. Inkubieren bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit für 14 bis 28 Tage 50% der Wachstumsmedien alle 7 Tage ersetzt werden . </li></ol> 7. adhärenten Kultur von neuralen Vorläuferzellen <ol><li> Um ein ausreichendes Volumen von 0,1% igen Gelatinelösung, die Oberfläche einer Gewebekulturschale und Heilung in einem Inkubator bei 37 ° C für 1 h vor der Verarbeitung des Neurosphären für adhärente Kultur zu decken. </li><li> Von der schwimmenden Neurosphären Kultur, verbinden alle Medien in einem einzigen Rohr. Pellet die neurospheres durch Zentrifugation bei 100 xg für 7 min und den Überstand entfernen. </li><li> Resuspendieren des Pellets in 1 ml 0,1% Trypsin EDTA und Inkubation bei 37 ° C für 7 min in einem Wasserbad. Pipette vorsichtig auf und ab 100 Mal, mit einer 200 ul Pipettenspitze. </li><li> 5 ml Serum-Medien für 7 min, um die Enzymreaktion und Zentrifuge bei 100 xg zu stoppen. Überstand verwerfen und Zellpellet in 1 ml komplettem Wachstumsmedium und zählen die Anzahl der lebenden Zellen Trypanblaufarbstoff Ausschluss Verwendung und einer Zählkammer. </li><li> Verdünnen Sie die Zellsuspension in komplettem Wachstumsmedium, entfernen Sie die Gelatine aus den Gewebekulturflaschen und Samen der Zellen bei 1 x 10 4 Zellen / cm 2. </li><li> Inkubieren der Zellen bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit. Ersetzen Sie die Medien mit frischen, erwärmt, vollständige Wachstumsmedien alle drei Tage, bis die Kultur etwa 80% Konfluenz erreicht (nach ca. 7 Tage). </li></ol> 8. Neural Colony Forming Assay <ol><li> Kombinieren Neural Colony Forming Zelle (NCFC) Assay Medienkomponenten: NCFC serumfreien Medium, Proliferation NS-A und Kollagenlösung (3 mg / ml). Nachtrag: Dieses Medium mit 2 ug / ml Heparin, 20 ng / ml EGF und 10 ng / ml bFGF. </li><li> Hippokampus-Gewebedissoziation Nach Resuspendieren der Zellen in neurale koloniebildenden Assay-Medium, vorgewärmt auf 37 ° C in einem Wasserbad. Samen der Zellen bei einer klonalen Dichte von 1,1 x 10 5 Zellen / ml in eine 35 mm Kulturschale. </li><li> Inkubieren der Zellen in diesem halbfesten Kollagen - Matrix bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit. Kultur für 28 Tage, 50% des Wachstumsmediums alle 7 Tage ersetzt werden. </li></ol> 9. Differenzierung von neuralen Vorläuferzellen <ol><li> Um ein ausreichendes Volumen von 5 &amp; mgr; g / cm 2 Laminin Lösung , um die Oberfläche einer Gewebekulturschale und Heilung in einem Inkubator bei 37 ° C für 24 h zu decken , bevor die Zellen für die Verarbeitung differentiation. Hinweis: Entfernen Sie die Laminin - Lösung und spülen Sie die beschichtete Oberfläche zweimal in DPBS unmittelbar vor der Aussaat. </li><li> Wenn Passagierung Saatgut der Zellsuspension mit 1 x 10 4 Zellen / cm 2 in DMEM-F12 (3: 1) Medium (vorgewärmt auf 37 ° C) mit 20 ng / ml EGF und 40 ng / ml bFGF auf die Laminin beschichteten Kulturschale. </li><li> Inkubieren der Zellen bei 37 ° C, 5% CO 2 und&gt; 95% Luftfeuchtigkeit und erlauben Zellen für 3 Tage zu vermehren. </li><li> Um die anhaftende neurale Vorläuferzellen differenzieren, nach 3 Tagen das Medium mit Differenzierungsmedium, das DMEM-F12 ersetzen (3: 1) Medium vorgewärmt auf 37 ° C, supplementiert mit 10 ng / ml brain derived neurotrophic factor (BDNF). </li><li> alle 3 Tage während der Differenzierung 50% der Medien zu ersetzen. Die Differenzierung erfolgt schrittweise über etwa 21 bis 28 Tagen haben. Achtung: Sobald die Zellen zu differenzieren begonnen, ersetzen nur 50% der Medien an einem time als Zutritt von Luft kann eine nachteilige Wirkung auf die Gesundheit der Zellen haben. </li></ol>

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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=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>

The authors have nothing to disclose.

Die Protokolle werden hier beschrieben, optimiert für die Maximierung der Zelllebensfähigkeit günstige Kulturbedingungen zu halten. Die Geschwindigkeit und die Sorgfalt bei der Extraktion, Isolierung und Expansion von entscheidender Bedeutung. Ein entscheidender Schritt für adhärenten einschichtigen Expansion Gründung ist die effektive Spaltung der primären Neurosphären. Nach Durchgang distanzierte unzureichend Sekundär schwebenden Neurosphären Neurosphären erzeugen kann. Während Medienwechsel können diese ...

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>
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		<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>
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		<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>
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			<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>
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		<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>
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			<td height="21" style="height:21px;">40 &#181;m cell strainer</td>
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			<td style="width:252px;">352340</td>
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			<td height="21" style="height:21px;">6 well culture plate</td>
			<td style="width:196px;">BD Biosciences</td>
			<td style="width:252px;">351146</td>
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			<td height="21" style="height:21px;width:337px;">B-27 Supplement (50X) serum free</td>
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			<td style="width:252px;">17504044</td>
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			<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>
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			<td height="21" style="height:21px;">Brain derived neurotrophic factor (BDNF)</td>
			<td style="width:196px;">Millipore</td>
			<td style="width:252px;">GF029</td>
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			<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>
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			<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>
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			<td height="21" style="height:21px;width:337px;">DPBS</td>
			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">14190250</td>
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		<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>
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		<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>
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		<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>
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		<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>
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			<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>
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		<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>
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		<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>
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			<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>
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			<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>
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			<td height="21" style="height:21px;">Povidone-iodine</td>
			<td style="width:196px;">Munipharma</td>
			<td style="width:252px;">Betadine</td>
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			<td style="width:196px;">Life Technologies</td>
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			<td style="width:196px;">Life Technologies</td>
			<td style="width:252px;">25200056</td>
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			<td style="width:196px;">ThermoFisher Scientific</td>
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			<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>
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			<td height="21" style="height:21px;width:337px;">Centrifuge</td>
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			<td style="width:252px;">Universal 320R</td>
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			<td height="21" style="height:21px;width:337px;">Manual pipettes</td>
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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.

Durch die Verwendung von in vitro - Assays neuralen Vorläufer, Neurogenese und Populationen neuralen Vorläuferzelle wurden über die dorsoventral Achse des erwachsenen Hippokampus canine charakterisiert und verglichen. Neurale Vorläuferzellen aus isolierten Hippocampus-Gewebe abgeleitet gebildet Floating Neurosphären innerhalb von 14 Tagen der Isolation, die einen Durchmesser von 100 &amp; mgr; m von 28 Tagen der Kultur zu erreichen. abgeleiteten Neurosphären aus dorsalen u...

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

Der Eckzahn Gehirn ist ein wertvolles Modell, bei dem die adulte Neurogenese zu studieren. Präsentiert hier sind Protokolle zur Isolierung und erweitert erwachsenen Hunde-Hippocampus-neuralen Vorläuferzellen aus primären Hirngewebe.

Isolierung und Expansion von adulten Canine Hippocampus Neural Precursors

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

isolation-and-expansion-of-adult-canine-hippocampal-neural-precursors

Research

JoVE Journal

Developmental Biology

7.0K Aufrufe.  University of Sydney.  Der Eckzahn Gehirn ist ein wertvolles Modell, bei dem die adulte Neurogenese zu studieren. Präsentiert hier sind Protokolle zur Isolierung und erweitert erwachsenen Hunde-Hippocampus-neuralen Vorläuferzellen aus primären Hirngewebe. 

Serie: Isolation and Expansion of Adult Canine Hippocampal Neural Precursors

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

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.

The adult mammalian spinal cord contains neural stem/progenitor cells (NSPCs) that can be isolated and expanded in culture. This protocol describes the harvesting, isolation, culture, and passaging of NSPCs generated from the periventricular region of the adult spinal cord from the rat and from human organ transplant donors.

Isolierung von Neural Stamm- / Vorläuferzellen aus dem Periventrikuläre Region der erwachsenen Ratte und Human Spinal Cord

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

Forschung

Entwicklungsbiologie

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

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Stabile und effiziente genetische Veränderung von Zellen in der erwachsenen Maus V-SVZ für die Analyse von Neural Stem Cell Autonome und Nicht autonome Effekte

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

The cerebellar external granule layer (EGL) is the site of the largest transit amplification in the developing brain, and an excellent model for studying neuronal proliferation and differentiation. In addition, evolutionary modifications of its proliferative capability have been responsible for the dramatic expansion of cerebellar size in the amniotes, making the cerebellum an excellent model for evo-devo studies of the vertebrate brain. The constituent cells of the EGL, cerebellar granule progenitors, also represent a significant cell of origin for medulloblastoma, the most prevalent paediatric neuronal tumour. Following transit amplification, granule precursors migrate radially into the internal granular layer of the cerebellum where they represent the largest neuronal population in the mature mammalian brain. In chick, the peak of EGL proliferation occurs towards the end of the second week of gestation. In order to target genetic modification to this layer at the peak of proliferation, we have developed a method for genetic manipulation through ex vivo electroporation of cerebellum slices from embryonic Day 14 chick embryos. This method recapitulates several important aspects of in vivo granule neuron development and will be useful in generating a thorough understanding of cerebellar granule cell proliferation and differentiation, and thus of cerebellum development, evolution and disease.

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

The cerebellar external granule layer is the site of the largest transit amplification in the developing brain. Here, we present a protocol to target genetic modification to this layer at the peak of proliferation using ex vivo electroporation and culture of cerebellar slices from embryonic Day 14 chick embryos.

Ex-vivo-Kultur von Chick Kleinhirn-Slices und räumlich gezielte Elektroporation von Körnerzellvorstufen

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

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolierung und Kultur von Adult Zebrafisch brain-derived Neurosphären

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

This paper describes how to use a custom manufactured, commercially available enclosed cell culture system for basic and preclinical research. Biosafety cabinets (BSCs) and incubators have long been the standard for culturing and expanding cell lines for basic and preclinical research. However, as the focus of many stem cell laboratories shifts from basic research to clinical translation, additional requirements are needed of the cell culturing system. All processes must be well documented and have exceptional requirements for sterility and reproducibility. In traditional incubators, gas concentrations and temperatures widely fluctuate anytime the cells are removed for feeding, passaging, or other manipulations. Such interruptions contribute to an environment that is not the standard for cGMP and GLP guidelines. These interruptions must be minimized especially when cells are utilized for therapeutic purposes. The motivation to move from the standard BSC and incubator system to a closed system is that such interruptions can be made negligible. Closed systems provide a work space to feed and manipulate cell cultures and maintain them in a controlled environment where temperature and gas concentrations are consistent. This way, pluripotent and multipotent stem cells can be maintained at optimum health from the moment of their derivation all the way to their eventual use in therapy.

Here is a protocol to grow pluripotent stem cells (PSC) and neural stem cells (NSC) in an enclosed cell culture system that permits maximum sterility and reproducibility, replacing the traditional biosafety cabinet and incubator. This equipment meets clinical good manufacturing practice (cGMP) and clinical good lab practice (cGLP) guidelines.

Die Kultivierung humanen pluripotenten und Neurale Stammzellen in einem geschlossenen Zellkultursystem für Grund- und Präklinische Forschung

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

Mesenchymal stem/stromal cells (MSC) are promising candidates for use in cell-based therapies. In most cases, therapeutic response appears to be cell-dose dependent. Human term placenta is rich in MSC and is a physically large tissue that is generally discarded following birth. Placenta is an ideal starting material for the large-scale manufacture of multiple cell doses of allogeneic MSC. The placenta is a fetomaternal organ from which either fetal or maternal tissue can be isolated. This article describes the placental anatomy and procedure to dissect apart the decidua (maternal), chorionic villi (fetal), and chorionic plate (fetal) tissue. The protocol then outlines how to isolate MSC from each dissected tissue region, and provides representative analysis of expanded MSC derived from the respective tissue types. These methods are intended for pre-clinical MSC isolation, but have also been adapted for clinical manufacture of placental MSC for human therapeutic use.

Herein we describe methods for the dissection of fetal and maternal tissues from human term placenta, followed by isolation and expansion of mesenchymal stem/stromal cells (MSC) from these tissues.

Isolierung und Expansion von mesenchymalen Stamm- / Stroma-Zellen aus humanen Placenta Gewebe gewonnen

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

Notch signaling is a key component of multiple physiological and pathological processes. The nature of Notch signaling, however, makes in vitro investigation of its varying and sometimes contradictory roles a challenge. As a component of direct cell-cell communication with both receptors and ligands bound to the plasma membrane, Notch signaling cannot be activated in vitro by simple addition of ligands to culture media, as is possible with many other signaling pathways. Instead, Notch ligands must be presented to cells in an immobilized state. 
Variations in methods of Notch signaling activation can lead to different outcomes in cultured cells. In osteoclast precursors, in particular, differences in methods of Notch stimulation and osteoclast precursor culture and differentiation have led to disagreement over whether Notch signaling is a positive or negative regulator of osteoclast differentiation. While closer comparisons of osteoclast differentiation under different Notch stimulation conditions in vitro and genetic models have largely resolved the controversy regarding Notch signaling and osteoclasts, standardized methods of continuous and temporary stimulation of Notch signaling in cultured cells could prevent such discrepancies in the future.
This protocol describes two methods for stimulating Notch signaling specifically in cultured mouse osteoclast precursors, though these methods should be applicable to any adherent cell type with minor adjustments. The first method produces continuous stimulation of Notch signaling and involves immobilizing Notch ligand to a tissue culture surface prior to the seeding of cells. The second, which uses Notch ligand bound to agarose beads allows for temporary stimulation of Notch signaling in cells that are already adhered to a culture surface. This protocol also includes methods for detecting Notch activation in osteoclast precursors as well as representative transcriptional markers of Notch signaling activation.

Notch signaling is a form of cellular communication that relies upon direct contact between cells. To properly induce Notch signaling in vitro, Notch ligands must be presented to cells in an immobilized state. This protocol describes methods for in vitro stimulation of Notch signaling in mouse osteoclast precursors.

Die Stimulation der Notch-Signalweg in Maus-Osteoklasten Precursors

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

The epicardium, an epithelial cell layer covering the myocardium, has an essential role during cardiac development, as well as in the repair response of the heart after ischemic injury. When activated, epicardial cells undergo a process known as epithelial to mesenchymal transition (EMT) to provide cells to the regenerating myocardium. Furthermore, the epicardium contributes via secretion of essential paracrine factors. To fully appreciate the regenerative potential of the epicardium, a human cell model is required. Here we outline a novel cell culture model to derive primary epicardial derived cells (EPDCs) from human adult and fetal cardiac tissue. To isolate EPDCs, the epicardium is dissected from the outside of the heart specimen and processed into a single cell suspension. Next, EPDCs are plated and cultured in EPDC medium containing the ALK 5-kinase inhibitor SB431542 to maintain their epithelial phenotype. EMT is induced by stimulation with TGF&#946;. This method enables, for the first time, the study of the process of human epicardial EMT in a controlled setting, and facilitates gaining more insight in the secretome of EPDCs that may aid heart regeneration. Furthermore, this uniform approach allows for direct comparison of human adult and fetal epicardial behavior.

The epicardium plays a crucial role in the development and repair of the heart by providing cells and growth factors to the myocardial wall. Here, we describe a method to culture human primary epicardial cells that enables the study and comparison of their developmental and adult characteristics.

Die Isolation und die Kultur der primären Epicardial Zellen aus menschlichen Erwachsenen und fetalen Herzens Exemplare

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

Interest in mesenchymal stem cells (MSCs) has increased over the past decade due to their ease of isolation, expansion, and culture. Recently, studies have demonstrated the wide differentiation capacity that these cells possess. The ovary represents a promising candidate for cell-based therapies due to the fact that it is rich in MSCs and that it is frequently discarded after ovariectomy surgeries as biological waste. This article describes procedures for the isolation, expansion, and differentiation of MSCs derived from the canine ovary, without the necessity of cell-sorting techniques. This protocol represents an important tool for regenerative medicine because of the broad applicability of these highly differentiable cells in clinical trials and therapeutic uses.

Herein, we describe a method for the isolation, expansion, and differentiation of mesenchymal stem cells from canine ovarian tissue.

Ableitung und Differenzierung von Eckzahn Eierstock mesenchymalen Stammzellen

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

Real-time Live-cell Flow Cytometry to Investigate Calcium Influx, Pore Formation, and Phagocytosis by P2X7 Receptors in Adult Neural Progenitor Cells

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a method whereby live-cell flow cytometry is extended upon to analyze the multiple functions of P2X7 receptor activation in real-time. Using a time module installed on a flow cytometer, live-cell functionality can be assessed and plotted over a given time period to explore the kinetics of calcium influx, transmembrane pore formation, and phagocytosis. This simple method is advantageous as all three canonical functions of the P2X7 receptor can be assessed using one machine, and the gathered data plotted over time provides information on the entire live-cell population rather than single-cell recordings often obtained using technically challenging patch-clamp methods. Calcium influx experiments use a calcium indicator dye, while P2X7 pore formation assays rely on ethidium bromide being allowed to pass through the transmembrane pore formed upon high agonist concentrations. Yellow-green (YG) latex beads are utilized to measure phagocytosis. Specific agonists and antagonists are applied to investigate the effects of P2X7 receptor activity. Individually, these methods can be modified to provide quantitative data on any number of calcium channels and purinergic and scavenger receptors. Taken together, they highlight how the use of real-time live-cell flow cytometry is a rapid, cost-effective, reproducible, and quantifiable method to investigate P2X7 receptor function.

Providing single-cell sensitivity, real-time flow cytometry is uniquely suited to quantify multimodal receptor functions of live cultures. Using adult neural progenitor cells, the P2X7 receptor function was assessed via calcium influx detected by calcium indicator dye, transmembrane pore formation by ethidium bromide uptake, and phagocytosis using fluorescent latex beads.

Echtzeit Live-Zelle Durchflusszytometrie, Calcium Zustrom, Porenbildung und Phagozytose durch P2X7-Rezeptoren im Erwachsenen neurale Vorläuferzellen zu untersuchen

Live-cell flow cytometry is increasingly used among cell biologists to quantify biological processes in a living cell culture. This protocol describes a ...