Name: obtaining human microglia from adult human brain tissue
Uploaded: 2020-08-30T15:00:00
Description: Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

Das Labor des SJ wurde mit institutionellen Zuschüssen des IITJ eingerichtet und wird durch Zuschüsse der Abteilung Biotechnologie (BT/PR12831/MED/30/1489/2015) und des Ministeriums für Elektronik und Informationstechnologie der indischen Regierung (Nr. 4(16)/2019-ITEA) finanziert. Die menschlichen Hirngewebeabschnitte wurden vom All India Institute of Medical Sciences (AIIMS) Jodhpur nach der Freigabe der institutionellen Ethikkommission bezogen. Wir danken Mayank Rathor, B.Tech Student Mitglied der Design and Arts Society IIT Jodhpur, für die Videofilmunterstützung.

Alle Gewebe wurden nach ethischer Freigabe von den Ethikkommissionen des Indian Institute of Technology Jodhpur und all India Institute of Medical Sciences (AIIMS) Jodhpur erworben.
1.Gewebeerwerb und -verarbeitung (Tag 0)
<ol><li>Sammeln Sie das Gewebe in einem 50 ml Rohr mit 10 ml eiskalte künstliche Zerebrospinalflüssigkeit (aCSF) (2 mM CaCl22 H2O, 10 mM Glukose, 3 mM KCl, 26 mM NaHCO3, 2,5 mM NaH2PO4, 1 mM MgCl2-H2<...

<ol>
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J., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+the+TBI+brain:+The+good,+the+bad,+and+the+dysregulated.">Microglia in the TBI brain: The good, the bad, and the dysregulated.</a> Experimental Neurology. 275 (03), 316-327 (2016).</li><li>Inoue, K., Tsuda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+in+neuropathic+pain:+cellular+and+molecular+mechanisms+and+therapeutic+potential.">Microglia in neuropathic pain: cellular and molecular mechanisms and therapeutic potential.</a> Nature Reviews Neuroscience. 19 (3), 138-152 (2018).</li><li>Bellver-Landete, V., 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=Microglia+are+an+essential+component+of+the+neuroprotective+scar+that+forms+after+spinal+cord+injury.">Microglia are an essential component of the neuroprotective scar that forms after spinal cord injury.</a> Nature Communications. 10 (1), 518 (2019).</li><li>Gutmann, D. H., Kettenmann, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia/Brain+Macrophages+as+Central+Drivers+of+Brain+Tumor+Pathobiology.">Microglia/Brain Macrophages as Central Drivers of Brain Tumor Pathobiology.</a> Neuron. 104 (3), 442-449 (2019).</li><li>Smith, A. M., Dragunow, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+human+side+of+microglia.">The human side of microglia.</a> Trends in Neurosciences. 37 (3), 125-135 (2014).</li><li>Galatro, T. F., 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=Transcriptomic+analysis+of+purified+human+cortical+microglia+reveals+age-associated+changes.">Transcriptomic analysis of purified human cortical microglia reveals age-associated changes.</a> Nature Neuroscience. 20 (8), 1162-1171 (2017).</li><li>Friedman, B. 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=Diverse+Brain+Myeloid+Expression+Profiles+Reveal+Distinct+Microglial+Activation+States+and+Aspects+of+Alzheimer's+Disease+Not+Evident+in+Mouse+Models.">Diverse Brain Myeloid Expression Profiles Reveal Distinct Microglial Activation States and Aspects of Alzheimer's Disease Not Evident in Mouse Models.</a> Cell Reports. 22 (3), 832-847 (2018).</li><li>Rustenhoven, 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=PU.1+regulates+Alzheimer's+disease-associated+genes+in+primary+human+microglia.">PU.1 regulates Alzheimer's disease-associated genes in primary human microglia.</a> Molecular Neurodegeneration. 13 (1), 44 (2018).</li><li>Sierra, A., Gottfried-Blackmore, A. C., McEwen, B. S., Bulloch, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microglia+derived+from+aging+mice+exhibit+an+altered+inflammatory+profile.">Microglia derived from aging mice exhibit an altered inflammatory profile.</a> Glia. 55 (4), 412-424 (2007).</li><li>Mizee, M. R., 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+of+primary+microglia+from+the+human+post-mortem+brain:+effects+of+ante-+and+post-mortem+variables.">Isolation of primary microglia from the human post-mortem brain: effects of ante- and post-mortem variables.</a> Acta Neuropathologica Communications. 5 (1), 16 (2017).</li><li>Rustenhoven, 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=Isolation+of+highly+enriched+primary+human+microglia+for+functional+studies.">Isolation of highly enriched primary human microglia for functional studies.</a> Scientific Reports. 6 (1), 19371 (2016).</li><li>Spaethling, J. M., 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=Primary+Cell+Culture+of+Live+Neurosurgically+Resected+Aged+Adult+Human+Brain+Cells+and+Single+Cell+Transcriptomics.">Primary Cell Culture of Live Neurosurgically Resected Aged Adult Human Brain Cells and Single Cell Transcriptomics.</a> Cell Reports. 18 (3), 791-803 (2017).</li><li>Olah, M., 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=An+optimized+protocol+for+the+acute+isolation+of+human+microglia+from+autopsy+brain+samples.">An optimized protocol for the acute isolation of human microglia from autopsy brain samples.</a> Glia. 60 (1), 96-111 (2012).</li><li>Jha, 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=The+Inflammasome+Sensor,+NLRP3,+Regulates+CNS+Inflammation+and+Demyelination+via+Caspase-1+and+Interleukin-18.">The Inflammasome Sensor, NLRP3, Regulates CNS Inflammation and Demyelination via Caspase-1 and Interleukin-18.</a> The Journal of Neuroscience. 30 (47), 15811 (2010).</li><li>Freeman, 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=NLR+members+NLRC4+and+NLRP3+mediate+sterile+inflammasome+activation+in+microglia+and+astrocytes.">NLR members NLRC4 and NLRP3 mediate sterile inflammasome activation in microglia and astrocytes.</a> Journal of Experimental Medicine. 214 (5), 1351-1370 (2017).</li><li>Plant, S. 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Mikroglia sorgen für Homöostase im normalen Gehirn und spielen eine zentrale Rolle in der Pathophysiologie verschiedener neurologischer Erkrankungen4. Mikroglia sind zentral für die Neuroentwicklung und Bildung von Synapsen2. Mikroglia-Studien haben sich als entscheidend für das Verständnis der Entwicklung und des Fortschreitens verschiedener neurologischer Erkrankungen erwiesen4. Nagetier-Mikroglia sind das vorherrschende Modell der Wahl für p...

Das Zentralnervensystem (ZNS) besteht aus einem komplexen Netzwerk von Neuronen und Gliazellen1. Unter den Gliazellen fungieren Mikroglia als angeborene Immunzellen der ZNS2,3. Microglia sind verantwortlich für die Aufrechterhaltung der Homöostase im gesunden ZNS4. Mikroglia spielen auch eine wichtige Rolle in der Neuroentwicklung, durch den Schnitt von Synapsen2. Mikroglia sind zentral für die Pathophysiologie mehrerer neurologischer Erkrankungen, einschließlich, aber nicht beschränkt auf; Alzheimer-Krankheit5, Parkinson-Krankheit6, Schlaganfall7, Multiple Sklerose8, traumatische Hirnverletzung9, neuropathische Schmerzen10, Rückenmarksverletzung11 und Hirntumoren wie Gliome12.
Studien im Zusammenhang mit ZNS-Homöostase und Krankheiten nutzen Nagetier-Mikroglia aufgrund eines Mangels an kosteneffizienten und zeiteffizienten menschlichen primären Mikroglia-Isolationsprotokollen13. Nagetier-Mikroglia ähneln primären menschlichen Mikroglia in Der Expression von Genen wie Iba-1, PU.1, DAP12 und M-CSF-Rezeptor und haben wirksam beim Verständnis normaler sowie kranker Gehirn13. Interessanterweise variiert die Expression mehrerer immunbezogener Gene wie TLR4, MHC II, Siglec-11 und Siglec-3 zwischen menschlicher und Nagetier-Mikroglia13. Die Expression mehrerer Gene variiert auch in der zeitlichen Expression und bei neurodegenerativen Erkrankungen bei beiden Arten14,15. Diese signifikanten Unterschiede machen menschliche Mikroglia zu einem wesentlichen Modell, um die Mikrogliafunktion bei Homöostase und Krankheit zu untersuchen. Primäre menschliche Mikroglia kann auch ein wirksames Werkzeug für das präklinische Screening von potenziellen Wirkstoffkandidaten16sein. Die oben genannten Gründe unterstreichen den wachsenden Bedarf an kostengünstigen Protokollen zur Isolierung primärer menschlicher Mikroglia.
Wir haben ein Protokoll zur Isolierung von primären menschlichen Mikroglia aus erwachsenen menschlichen Hirngewebe als Ergebnis der chirurgischen Fenster für Tumorresektionen oder andere chirurgische Resektionen erstellt entwickelt. Die Methode unterscheidet sich hier erheblich von den bestehenden Methoden. Wir waren in der Lage, Mikroglia nach einer Transitzeit von etwa 75 Minuten von der Gewebesammelstelle bis zum Start des Isolationsprotokolls im Labor zu isolieren und zu kultiieren. Wir haben den Überstand von L929-Fibroblastenzellen verwendet, um das Wachstum isolierter Mikroglia zu fördern. Diese Methode konzentriert sich speziell auf die Kultur und Entwicklung von nur primären Mikroglia. Die resultierende Kultur vorbereitet ist etwa 80% Mikroglia. Während andere Protokolle eine angereicherte Kultur der Mikroglia durch Dichtegradientzentrifugation, Durchflusszytometrie und magnetische Perlen bieten, ist das Protokoll eine schnelle, einfache, robuste und kostengünstige Möglichkeit, primäre menschliche Mikroglia17,18,19,20zu kulturieren. Die Fähigkeit, chirurgisch entferntes lebendes erwachsenes Hirngewebe anstelle von festem Hirngewebe von Kadavern zu nutzen, erweist sich als zusätzlicher Vorteil dieser Methode im Gegensatz zu bestehenden Verfahren18,21.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibiotic-Antimycotic solution</td>
			<td>Himedia</td>
			<td>A002</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium chloride</td>
			<td>Sigma</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge (4 &#176;C)</td>
			<td>Sigma</td>
			<td>146532</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge tubes</td>
			<td>Abdos</td>
			<td>P10203</td>
			<td></td>
		</tr>
		<tr>
			<td>CO2 incubator</td>
			<td>New Brunswik</td>
			<td>Galaxy 170 S</td>
			<td></td>
		</tr>
		<tr>
			<td>D-Glucose</td>
			<td>Himedia</td>
			<td>GRM077</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM medium with glutamine</td>
			<td>Himedia</td>
			<td>AL007S</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Himedia</td>
			<td>RM9955</td>
			<td></td>
		</tr>
		<tr>
			<td>Flacon tube (50 ml)</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>50CD1058</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescein Ricinus communis agglutinin-1</td>
			<td>Vector</td>
			<td>FL-1081</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluorescent microscope</td>
			<td>Leica</td>
			<td>DM2000LED</td>
			<td></td>
		</tr>
		<tr>
			<td>Fluoroshield with DAPI</td>
			<td>Sigma</td>
			<td>F6057</td>
			<td></td>
		</tr>
		<tr>
			<td>GFAP antibody</td>
			<td>GA5</td>
			<td>3670S</td>
			<td></td>
		</tr>
		<tr>
			<td>Incubator shaker</td>
			<td>New Brunswik Scientific</td>
			<td>Innova 42</td>
			<td></td>
		</tr>
		<tr>
			<td>L929 cell line</td>
			<td>ATCC</td>
			<td>NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC&#160;CCL-1)</td>
			<td></td>
		</tr>
		<tr>
			<td>Laminar air flow</td>
			<td>Thermo Fsiher Scientific&#160;</td>
			<td>1386</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Himedia</td>
			<td>MB040</td>
			<td></td>
		</tr>
		<tr>
			<td>Monosodium phosphate</td>
			<td>Merck</td>
			<td>567545</td>
			<td></td>
		</tr>
		<tr>
			<td>Nutrient Mixture F-12 Ham Medium</td>
			<td>Himedia</td>
			<td>Al106S</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dish</td>
			<td>Duran Group</td>
			<td>237554805</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate buffered saline</td>
			<td>Himedia</td>
			<td>ML023</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>Himedia</td>
			<td>MB043</td>
			<td></td>
		</tr>
		<tr>
			<td>Serological pipette</td>
			<td>Labware</td>
			<td>LW-SP1010</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium bicarbonate</td>
			<td>Himedia</td>
			<td>MB045</td>
			<td></td>
		</tr>
		<tr>
			<td>Sucrose</td>
			<td>Himedia</td>
			<td>MB025</td>
			<td></td>
		</tr>
		<tr>
			<td>Syringe filter (0.2&#956;, 25 mm diameter)</td>
			<td>Axiva</td>
			<td>SFPV25R</td>
			<td></td>
		</tr>
		<tr>
			<td>T-25 tissue culture flasks suitable for adherent cell culture.</td>
			<td>Himedia</td>
			<td>TCG4-20X10NO</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA (0.25%)</td>
			<td>Gibco&#160;</td>
			<td>25200-056</td>
			<td></td>
		</tr>
	</tbody>
</table>

obtaining human microglia from adult human brain tissue

Mikroglia sind ansässige angeborene Immunzellen des Zentralnervensystems (ZNS). Mikroglia spielen eine entscheidende Rolle bei der Entwicklung, bei der Aufrechterhaltung der Homöostase und bei Infektionen oder Verletzungen. Mehrere unabhängige Forschungsgruppen haben die zentrale Rolle hervorgehoben, die Mikroglia bei Autoimmunerkrankungen, autoinflammatorischen Syndromen und Krebserkrankungen spielt. Die Aktivierung von Mikroglia bei einigen neurologischen Erkrankungen kann direkt an pathogenen Prozessen beteiligt sein. Primäre Mikroglia sind ein leistungsfähiges Werkzeug, um die Immunantworten im Gehirn, Zell-Zell-Wechselwirkungen und dysregulierte Mikroglia-Phänotypen bei Krankheiten zu verstehen. Primäre Mikroglia imitieren in vivo mikroglia-Eigenschaften besser als verewigte mikrogliarische Zelllinien. Menschliche Erwachsene Mikroglia weisen unterschiedliche Eigenschaften im Vergleich zu menschlichen fetalen und Nagetier Mikroglia. Dieses Protokoll bietet eine effiziente Methode zur Isolierung von primären Mikroglia aus erwachsenen menschlichen Gehirn. Das Studium dieser Mikroglia kann kritische Einblicke in Zell-Zell-Wechselwirkungen zwischen Mikroglia und anderen ansässigen Zellpopulationen im ZNS liefern, einschließlich Oligodendrozyten, Neuronen und Astrozyten. Darüber hinaus können Mikroglia aus verschiedenen menschlichen Gehirnen für die Charakterisierung einzigartiger Immunantworten für personalisierte Medizin und eine Vielzahl von therapeutischen Anwendungen kultiviert werden.

Durch die Verwendung des oben genannten Protokolls (Abbildung 1) konnten wir primäre menschliche Mikroglia aus lebendem chirurgisch resektiertem Hirngewebe isolieren. Kultivierte Zellen wurden mit Ricinus communis agglutinin-1 (RCA-1) Lektin für Mikroglia (grün) und mit Glial fibrilläres saures Protein (GFAP) für Astrozyten (rot)(Abbildung 2) gefärbt, wie zuvor beschrieben22,<sup ...

Watch this Scientific Journal Video about Obtaining Human Microglia from Adult Human Brain Tissue at JoVE.com

Obtaining Human Microglia from Adult Human Brain Tissue

Dieses Protokoll ist eine effiziente, kostengünstige und robuste Methode zur Isolierung primärer Mikroglia aus lebendem, erwachsenem, menschlichem Hirngewebe. Isolierte primäre menschliche Mikroglia kann als Werkzeug für die Untersuchung zellulärer Prozesse bei Homöostase und Krankheit dienen.

Gewinnung menschlicher Mikroglia aus erwachsenem menschlichem Hirngewebe

Microglia are resident innate immune cells of the central nervous system (CNS). Microglia play a critical role during development, in maintaining ...

Das übergeordnete Ziel des Protokolls ist es, primäre Mikrogliazellen aus lebenden erwachsenen menschlichen Hirngeweben zu isolieren. In unserem Fall wurde dies als chirurgisches Fenster während der Operation gesammelt. Dies wird durch das zuerst sammeln des Gehirngewebes in eiskalten PBS erreicht, das Gewebe wird dann in 1-mm-Würfel kleine Stücke gewürfelt, und ich habe gerade mit Hilfe von Trypsin EDTA.Danach werden die Zellen durch Zentrifugation geerntet und 48 Stunden lang in einem Kolben plattiert, der für die Androgenzellkultur geeignet ist. Die Zellen werden wieder aus dem Kolben geerntet und bis zur weiteren Verwendung kultiviert. Primäre menschliche Mikrogliazellen können nun durch Immunzytochemie charakterisiert und für weitere Experimente verwendet werden.Der Hauptvorteil unseres Protokolls zur Isolierung von Mikrogliazellen aus adulten menschlichen Hirngeweben besteht darin, dass es die primären adulten menschlichen Mikrogliazellen effektiv auf sehr kostengünstige Weise bereitstellt. Das Protokoll ist robust und reduziert den Zellverlust, indem es die Anzahl der Schritte reduziert, die in vorhandenen Protokollen verwendet werden. Sammeln Sie das Gewebe in einem 50-ml Falcon-Rohr mit 10 ml eiskaltem aCSF.Wischen Sie das Sammelrohr sorgfältig mit 70% Alkohol ab und übertragen Sie es in eine aseptische laminare Luftstromkammer. Entsorgen Sie das aCSF sorgfältig und wiegen Sie Gewebe in der Falcon-Röhre. Berechnen Sie das ungefähre Gewicht des Gewebes, indem Sie das Gewicht des leeren Falcon-Rohrs ableiten.Warm frisch aCSF bis 37 Grad Celsius. Halten Sie das Gewebe fünf Minuten lang in warmem ACSF auf. Dieser Schritt ist entscheidend, um den Zelltod zu vermeiden.Entsorgen Sie aCSF sorgfältig. Und waschen Gewebe einmal mit 1X PBS erwärmt auf 37 Grad Celsius. Stellen Sie sicher, dass das gesamte Blut bei Bedarf mit wiederholten PBS-Waschungen weggespült wird.Inkubieren Sie das Gewebe in warmen PBS für 5 Minuten. Entsorgen Sie die Hälfte der PBS sorgfältig. Übertragen Sie Gewebe zusammen mit den verbleibenden PBS in eine sterilisierte Petrischale.Entfernen Sie die verbleibendePBS sorgfältig mit 1-ml Pipette. Dies verhindert den Verlust von Gewebe. Würfel, die mit einem sterilen Skalpell in mindestens 1-mm-Würfel kleine Stücke würfeln.Fügen Sie 2 ml 0,25%Trypsin EDTA in die Petrischale und waschen Sie den Teller gründlich mit Hilfe von Pipette. Übertragen Sie das gewürfelte Gewebe in ein 50-ml Falcon-Rohr mit 10 ml pro Gramm Gewebe von 0,25%Trypsin EDTA. Mischen Sie durch Pipettieren durch eine 10-ml-Serologische Pipette.Inkubieren Sie die Röhre auf einem Shaker für 30 Minuten bei 37 Grad Celsius und der Geschwindigkeit von 250 U/min. Fügen Sie 10 ml neutralisierendes Medium hinzu, um Trypsin zu neutralisieren. Mit einer 10-ml-Serologischen Pipette gründlich mischen.Zentrifugieren Sie das Rohr bei 2000G bei 4 Grad Celsius für 10 Minuten. Entsorgen Sie den Überstand und setzen Sie das Pellet in 1 ml Kulturmedium wieder auf. Legen Sie die Zellen in einem T25-Kolben, der für Androgenzellen geeignet ist, und fügen Sie 4 ml zusätzliches Kulturmedium hinzu.Kulturmedium wird 20%L929 Überstand und 1%Streptomycin enthalten. Es wird empfohlen, L929 Überstand separat in Kolben hinzuzufügen, anstatt das Stoffkulturmedium hinzuzufügen. Wir sind mit dem Kolben fertig, um das Gewebe homogen zu entsorgen.Vermeiden Sie es, die Medien beim Schütteln an den Hals des Kolbens zu bringen, da dies die Wahrscheinlichkeit einer Kontamination erhöhen kann. Inkubieren Sie den Kolben bei 37 Grad Celsius mit 5% CO2 für 48 Stunden. Sammeln Sie die Medien aus T25 Kulturkolben am Tag 0 in Zentrifugenröhren vorbereitet.Zentrifuge die gesammelten Medien bei 1, 466G bei 4 Grad Celsius für 4 Minuten. Während die gesammelten Medien zentrifugiert werden, waschen Sie den Tag 0 Kolben einmal mit 1X PBS. Schütteln Sie den Kolben vorsichtig, um alle restlichen Gewebefragmente zu entfernen.Vermeiden Sie ein hartes Schütteln des Kolbens, da Restfragmente die Kultur nicht beeinträchtigen. Fügen Sie 5 ml frische Kulturmedien in den Kolben. Entsorgen Sie den Überstand aus zentrifugierten Rohren.1 ml Kulturmedium in eines der Rohre geben und gründlich mit Pipette mischen. Serial fügen Sie das Mischmedium mit Zellen zu anderen Röhren und mischen gründlich. Legen Sie die Zellen in einem separaten T25-Kolben, der für Androgenzellen geeignet ist.Fügen Sie 4 ml frische Kulturmedien in den Kolben. Inkubieren Sie beide Kolben bei 37 Grad Celsius mit 5% CO2 für 48 Stunden. Entsorgen Sie die Medien aus beiden Kolben, und fügen Sie neue 5 ml Kulturmedien hinzu.Inkubieren Sie die Kolben bei 37 Grad Celsius bei 5% CO2 für 48 Stunden. Am sechsten Tag werden die Zellen für weitere Experimente bereit sein. In den hier dargestellten Bildern.Erste Platte des Abschnitts A Astrozyten mit GFAP gebeizt, grün in der Farbe. Im zweiten Panel, Abschnitt A Mikroglia Fleck mit RCA, grün in der Farbe. Im Abschnitt B Mikroglia mit RCA gefärbt, die grün in der Farbe ist, und Astrozyten sind mit GFAP, rot in der Farbe gefärbt.Die Überlagerung der Bilder zeigt Mikroglia und Astrozyten zusammen. Aus den Bildern geht klar hervor, dass die Mehrheit der Zellen in der vorbereiteten Kultur Mikroglia sind. Die Bilder wurden durch geblendete Kontrolle quantifiziert und das Ergebnis wird in Abschnitt C dargestellt. Die Ergebnisse zeigten, dass etwa 80% der Zellen in der vorbereiteten Kultur Mikrogliazellen sind.Diese Technik kann in etwa eineinhalb Stunden durchgeführt werden. Nach diesem Verfahren sollte ein gutes Verständnis dafür haben, wie ich Grundierung Mikroglia aus erwachsenen menschlichen Gehirngeweben, die weiter für nachgelagerte Experimente verwendet werden kann wählen.

obtaining-human-microglia-from-adult-human-brain-tissue

Research

JoVE Journal

Neuroscience

Combining Computer Game-Based Behavioural Experiments With High-Density EEG and Infrared Gaze Tracking

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they yield data relevant to the cognitive processing that occurs under ecologically valid conditions. These two goals often are at odds, since well controlled stimuli often are too repetitive to sustain subjects' motivation. Studies employing electroencephalography (EEG) are often especially sensitive to this dilemma between ecological validity and experimental control: attaining sufficient signal-to-noise in physiological averages demands large numbers of repeated trials within lengthy recording sessions, limiting the subject pool to individuals with the ability and patience to perform a set task over and over again. This constraint severely limits researchers' ability to investigate younger populations as well as clinical populations associated with heightened anxiety or attentional abnormalities. Even adult, non-clinical subjects may not be able to achieve their typical levels of performance or cognitive engagement: an unmotivated subject for whom an experimental task is little more than a chore is not the same, behaviourally, cognitively, or neurally, as a subject who is intrinsically motivated and engaged with the task. A growing body of literature demonstrates that embedding experiments within video games may provide a way between the horns of this dilemma between experimental control and ecological validity. The narrative of a game provides a more realistic context in which tasks occur, enhancing their ecological validity (Chaytor &amp; Schmitter-Edgecombe, 2003). Moreover, this context provides motivation to complete tasks. In our game, subjects perform various missions to collect resources, fend off pirates, intercept communications or facilitate diplomatic relations. In so doing, they also perform an array of cognitive tasks, including a Posner attention-shifting paradigm (Posner, 1980), a go/no-go test of motor inhibition, a psychophysical motion coherence threshold task, the Embedded Figures Test (Witkin, 1950, 1954) and a theory-of-mind (Wimmer &amp; Perner, 1983) task. The game software automatically registers game stimuli and subjects' actions and responses in a log file, and sends event codes to synchronise with physiological data recorders. Thus the game can be combined with physiological measures such as EEG or fMRI, and with moment-to-moment tracking of gaze. Gaze tracking can verify subjects' compliance with behavioural tasks (e.g. fixation) and overt attention to experimental stimuli, and also physiological arousal as reflected in pupil dilation (Bradley et al., 2008). At great enough sampling frequencies, gaze tracking may also help assess covert attention as reflected in microsaccades - eye movements that are too small to foveate a new object, but are as rapid in onset and have the same relationship between angular distance and peak velocity as do saccades that traverse greater distances. The distribution of directions of microsaccades correlates with the (otherwise) covert direction of attention (Hafed &amp; Clark, 2002).

Procedures for recording high-density EEG and gaze data during computer game-based cognitive tasks are described. Using a video game to present cognitive tasks enhances ecological validity without sacrificing experimental control.

Die Kombination Computer Game-Based Behavioural Experimente mit High-Density EEG-und Infrarot-Tracking-Gaze

Experimental paradigms are valuable insofar as the timing and other parameters of their stimuli are well specified and controlled, and insofar as they ...

Forschung

Neurowissenschaften

A Rapid Approach to High-Resolution Fluorescence Imaging in Semi-Thick Brain Slices

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are characteristic to neurons in both normal and diseased brain. Towards this, a great deal of effort has been placed on building high-resolution neuroanatomical maps1-3. With the expansion of molecular genetics and advances in light microscopy has come the ability to query not only neuronal morphologies, but also the molecular and cellular makeup of individual neurons and their associated networks4. Major advances in the ability to mark and manipulate neurons through transgenic and gene targeting technologies in the rodent now allow investigators to 'program' neuronal subsets at will5-6. Arguably, one of the most influential contributions to contemporary neuroscience has been the discovery and cloning of genes encoding fluorescent proteins (FPs) in marine invertebrates7-8, alongside their subsequent engineering to yield an ever-expanding toolbox of vital reporters9. Exploiting cell type-specific promoter activity to drive targeted FP expression in discrete neuronal populations now affords neuroanatomical investigation with genetic precision.
Engineering FP expression in neurons has vastly improved our understanding of brain structure and function. However, imaging individual neurons and their associated networks in deep brain tissues, or in three dimensions, has remained a challenge. Due to high lipid content, nervous tissue is rather opaque and exhibits auto fluorescence. These inherent biophysical properties make it difficult to visualize and image fluorescently labelled neurons at high resolution using standard epifluorescent or confocal microscopy beyond depths of tens of microns. To circumvent this challenge investigators often employ serial thin-section imaging and reconstruction methods10, or 2-photon laser scanning microscopy11. Current drawbacks to these approaches are the associated labor-intensive tissue preparation, or cost-prohibitive instrumentation respectively.
Here, we present a relatively rapid and simple method to visualize fluorescently labelled cells in fixed semi-thick mouse brain slices by optical clearing and imaging. In the attached protocol we describe the methods of: 1) fixing brain tissue in situ via intracardial perfusion, 2) dissection and removal of whole brain, 3) stationary brain embedding in agarose, 4) precision semi-thick slice preparation using new vibratome instrumentation, 5) clearing brain tissue through a glycerol gradient, and 6) mounting on glass slides for light microscopy and z-stack reconstruction (Figure 1).
For preparing brain slices we implemented a relatively new piece of instrumentation called the 'Compresstome' VF-200 (http://www.precisionary.com/products_vf200.html). This instrument is a semi-automated microtome equipped with a motorized advance and blade vibration system with features similar in function to other vibratomes. Unlike other vibratomes, the tissue to be sliced is mounted in an agarose plug within a stainless steel cylinder. The tissue is extruded at desired thicknesses from the cylinder, and cut by the forward advancing vibrating blade. The agarose plug/cylinder system allows for reproducible tissue mounting, alignment, and precision cutting. In our hands, the 'Compresstome' yields high quality tissue slices for electrophysiology, immunohistochemistry, and direct fixed-tissue mounting and imaging. Combined with optical clearing, here we demonstrate the preparation of semi-thick fixed brain slices for high-resolution fluorescent imaging.

Here we describe a rapid and simple method to image fluorescently labeled cells in semi-thick brain slices. By fixing, slicing, and optically clearing brain tissue we describe how standard epifluorescent or confocal imaging can be used to visualize individual cells and neuronal networks within intact nervous tissue.

Eine schnelle Annäherung an High-Resolution Fluoreszenz-Imaging in Semi-Thick Hirnschnitten

A fundamental goal to both basic and clinical neuroscience is to better understand the identities, molecular makeup, and patterns of connectivity that are ...

Fiber-optic Implantation for Chronic Optogenetic Stimulation of Brain Tissue

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard for analyzing patterns of synaptic connectivity, but paired electrophysiological recordings can be both cumbersome and experimentally limiting. The development of optogenetics has introduced an elegant method to stimulate neurons and circuits, both in vitro1 and in vivo2,3. By exploiting cell-type specific promoter activity to drive opsin expression in discrete neuronal populations, one can precisely stimulate genetically defined neuronal subtypes in distinct circuits4-6. Well described methods to stimulate neurons, including electrical stimulation and/or pharmacological manipulations, are often cell-type indiscriminate, invasive, and can damage surrounding tissues. These limitations could alter normal synaptic function and/or circuit behavior. In addition, due to the nature of the manipulation, the current methods are often acute and terminal. Optogenetics affords the ability to stimulate neurons in a relatively innocuous manner, and in genetically targeted neurons. The majority of studies involving in vivo optogenetics currently use a optical fiber guided through an implanted cannula6,7; however, limitations of this method include damaged brain tissue with repeated insertion of an optical fiber, and potential breakage of the fiber inside the cannula. Given the burgeoning field of optogenetics, a more reliable method of chronic stimulation is necessary to facilitate long-term studies with minimal collateral tissue damage. Here we provide our modified protocol as a video article to complement the method effectively and elegantly described in Sparta et al.8 for the fabrication of a fiber optic implant and its permanent fixation onto the cranium of anesthetized mice, as well as the assembly of the fiber optic coupler connecting the implant to a light source. The implant, connected with optical fibers to a solid-state laser, allows for an efficient method to chronically photostimulate functional neuronal circuitry with less tissue damage9 using small, detachable, tethers. Permanent fixation of the fiber optic implants provides consistent, long-term in vivo optogenetic studies of neuronal circuits in awake, behaving mice10 with minimal tissue damage.

The development of optogenetics now provides the means to precisely stimulate genetically defined neurons and circuits, both in vitro and in vivo. Here we describe the assembly and implantation of a fiber optic for chronic photostimulation of brain tissue.

LWL-Implantation für chronische optogenetische Stimulation des Hirngewebes

Elucidating patterns of neuronal connectivity has been a challenge for both clinical and basic neuroscience. Electrophysiology has been the gold standard ...

Simple Generation of a High Yield Culture of Induced Neurons from Human Adult Skin Fibroblasts

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily accessible. Through this route, mature neurons can be obtained in a matter of a few weeks. Here, we describe a straightforward and rapid one-step protocol to obtain iNs from dermal fibroblasts obtained through biopsy samples from adult human donors. We explain each step of the process, including the maintenance of the dermal fibroblasts, the freezing procedure to build a stock of the cell line, seeding of the cells for reprogramming, as well as the culture conditions during the conversion process. In addition, we describe the preparation of glass coverslips for electrophysiological recordings, long-term coating conditions, and fluorescence activated cell sorting (FACS). We also illustrate examples of the results to be expected. The protocol described here is easy to perform and can be applied to human fibroblasts derived from human skin biopsies from patients with various different diagnoses and ages. This protocol generates a sufficient amount of iNs which can be used for a wide array of biomedical applications, including disease modeling, drug screening, and target validation.

Direct neuronal reprogramming generates neurons that maintain the age of the starting somatic cell. Here, we describe a single vector-based method to generate induced neurons from dermal fibroblasts obtained from adult human donors.

Einfache Erstellung einer High-Yield-Kultur der induzierten Neuronen aus Menschenhaut Erwachsenen Fibroblasten

Induced neurons (iNs), the product of somatic cells directly converted to neurons, are a way to obtain patient-derived neurons from tissue that is easily ...

Isolation of Cerebral Capillaries from Fresh Human Brain Tissue

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic strategies that hold the promise to enhance brain drug delivery, improve brain protection, and treat brain disorders. However, studying the human blood-brain barrier function is challenging. Thus, there is a critical need for appropriate models. In this regard, brain capillaries isolated from human brain tissue represent a unique tool to study barrier function as close to the human in vivo situation as possible. Here, we describe an optimized protocol to isolate capillaries from human brain tissue at a high yield and with consistent quality and purity. Capillaries are isolated from fresh human brain tissue using mechanical homogenization, density-gradient centrifugation, and filtration. After the isolation, the human brain capillaries can be used for various applications including leakage assays, live cell imaging, and immune-based assays to study protein expression and function, enzyme activity, or intracellular signaling. Isolated human brain capillaries are a unique model to elucidate the regulation of the human blood-brain barrier function. This model can provide insights into central nervous system (CNS) pathogenesis, which will help the development of therapeutic strategies for treating CNS disorders.

Isolated brain capillaries from human brain tissue can be used as a preclinical model to study barrier function under physiological and pathophysiological conditions. Here, we present an optimized protocol to isolate brain capillaries from fresh human brain tissue.

Isolierung der zerebralen Kapillaren aus frischem menschlichen Hirngewebe

Understanding blood-brain barrier function under physiological and pathophysiological conditions is critical for the development of new therapeutic ...

Isolation and RNA Extraction of Neurons, Macrophages and Microglia from Larval Zebrafish Brains

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is important to isolate these cells without changing their gene expression profile. The zebrafish model provides a large number of transgenic fish lines in which specific cell types are labelled; for example neurons in the NBT:DsRed line or macrophages/microglia in the mpeg1:eGFP line. Furthermore, antibodies have been developed to stain specific cells, such as microglia with the 4C4 antibody.
Here, we describe the isolation of neurons, macrophages and microglia from larval zebrafish brains. Central to this protocol is the avoidance of an enzymatic tissue digestion at 37 &#176;C, which could modify cellular profiles. Instead a mechanical system of tissue homogenization at 4 &#176;C is used. This protocol entails homogenization of brains into cell suspension, their immuno-staining and the isolation of neurons, macrophages and microglia by FACS. Afterwards, we extracted RNA from those cells and evaluated their quality/quantity. We managed to obtain RNA of high quality (RNA Integrity Number (RIN) &#62; 7) to perform qPCR on macrophages/microglia and neurons, and transcriptomic analysis on microglia. This approach enables a better characterization of these cells, as well as a clearer understanding about their role in development and pathologies.

We present a protocol to isolate neurons, macrophages and microglia from larval zebrafish brains under physiological and pathological conditions. Upon isolation, RNA is extracted from these cells to analyze their gene expression profile. This protocol allows for the collection of high-quality RNA for performing downstream analysis like qPCR and transcriptomics.

Isolation und RNA-Extraktion von Neuronen, Makrophagen und Mikroglia aus Larven Zebrafisch Gehirne

To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...

Short-Term Free-Floating Slice Cultures from the Adult Human Brain

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of slice cultures in neuroscience, studies using adult nervous tissue to prepare such cultures are still scarce, particularly those from human subjects. The use of adult human tissue to prepare slice cultures is particularly attractive to enhance the understanding of human neuropathologies, as they hold unique properties typical of the mature human brain lacking in slices produced from rodent (usually neonatal) nervous tissue. This protocol describes how to use brain tissue collected from living human donors submitted to resective brain surgery to prepare short-term, free-floating slice cultures. Procedures to maintain and perform biochemical and cell biology assays using these cultures are also presented. Representative results demonstrate that the typical human cortical lamination is preserved in slices after 4 days in vitro (DIV4), with expected presence of the main neural cell types. Moreover, slices at DIV4 undergo robust cell death when challenged with a toxic stimulus (H2O2), indicating the potential of this model to serve as a platform in cell death assays. This method, a simpler and cost-effective alternative to the widely used protocol using membrane inserts, is mainly recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases. Finally, although the protocol is devoted to using cortical tissue collected from patients submitted to surgical treatment of pharmacoresistant temporal lobe epilepsy, it is argued that tissue collected from other brain regions/conditions should also be considered as sources to produce similar free-floating slice cultures.

A protocol to prepare free-floating slice cultures from adult human brain is presented. The protocol is a variation of the widely used slice culture method using membrane inserts. It is simple, cost-effective, and recommended for running short-term assays aimed to unravel mechanisms of neurodegeneration behind age-associated brain diseases.

Kurzfristig frei schwebende Scheibenkulturen aus dem erwachsenen menschlichen Gehirn

Organotypic, or slice cultures, have been widely employed to model aspects of the central nervous system functioning in vitro. Despite the potential of ...

Isolation of Region-specific Microglia from One Adult Mouse Brain Hemisphere for Deep Single-cell RNA Sequencing

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive human diseases. Considerable advances have been made to uncover the molecular signatures of homeostatic microglia as well as alterations of their gene expression in response to environmental stimuli. With the advent and maturation of single-cell genomic methodologies, it is increasingly recognized that heterogenous microglia may underlie the diverse roles they play in different developmental and pathological conditions. Further dissection of such heterogeneity can be achieved through efficient isolation of microglia from a given region of interest, followed by sensitive profiling of individual cells. Here, we provide a detailed protocol for the rapid isolation of microglia from different brain regions in a single adult mouse brain hemisphere. We also demonstrate how to use these sorted microglia for plate-based deep single-cell RNA sequencing. We discuss the adaptability of this method to other scenarios and provide guidelines for improving the system to accommodate large-scale studies.

We provide a protocol for isolation of microglia from different dissected regions of an adult mouse brain hemisphere, followed by semi-automated library preparation for deep single-cell RNA sequencing of full-length transcriptomes. This method will help to elucidate functional heterogeneity of microglia in health and disease.

Isolierung von regionsspezifischer Mikroglia von einer Erwachsenenmaus Gehirnhälfte für tiefe einzellige RNA-Sequenzierung

As resident macrophages in the central nervous system, microglia actively control brain development and homeostasis, and their dysfunctions may drive ...

Isolation of Adult Human Astrocyte Populations from Fresh-Frozen Cortex Using Fluorescence-Activated Nuclei Sorting

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular characterization. Fluorescence-activated nuclei sorting (FANS) can be used to successfully isolate and study human neuronal nuclei (NeuN+) populations from frozen archival tissue, thereby avoiding problems associated with handling&#160;fresh tissue. However, efforts to similarly isolate astroglia from the non-neuronal (NeuN-) element are lacking. A recently developed and validated immunotagging strategy uses three transcription factor antibodies to simultaneously isolate enriched neuronal (NeuN+), astrocyte (paired box protein 6 (PAX6)+NeuN-), and oligodendrocyte progenitor (OLIG2+NeuN-) nuclei populations from non-diseased, fresh (unfixed) snap-frozen&#160;postmortem human temporal neocortex tissue.
This technique was shown to be useful for the characterization of cell type-specific transcriptome alterations in primary pathological epilepsy neocortex. Transcriptomic analyses confirmed that PAX6+NeuN- sorted populations are robustly enriched for pan-astrocyte markers and capture astrocytes in both resting and reactive conditions. This paper describes the FANS methodology for the isolation of astrocyte-enriched nuclei populations from fresh-frozen human cortex, including tissue dissociation into single-nucleus (sn) suspension; immunotagging of nuclei with anti-NeuN and anti-PAX6 fluorescently conjugated antibodies; FANS gating strategies and quality control metrics for optimizing sensitivity and specificity during sorting and for confirming astrocyte enrichment; and recommended procurement for downstream transcriptome and chromatin accessibility sequencing at bulk or sn resolution. This protocol is applicable for non-necrotic, fresh-frozen, human cortical specimens with various pathologies and recommended postmortem tissue collection within 24 h.

We have developed a method that enriches for and isolates human astrocyte populations from fresh-frozen tissue for use in downstream molecular analyses.

Isolierung von erwachsenen menschlichen Astrozytenpopulationen aus frisch gefrorenen Kortex mittels fluoreszenzaktivierter Zellkernsortierung

The complexity of human astrocytes remains poorly defined in primary human tissue, requiring better tools for their isolation and molecular ...

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple neurological diseases. Studying human microglia in health and disease represents a challenge due to the extremely limited supply of human cells. Induced pluripotent stem cells (iPSCs) derived from human individuals can be used to circumvent this barrier. Here, it is demonstrated how to differentiate human iPSCs into microglia-like cells (iMGs) for in vitro experimentation. These iMGs exhibit the expected and physiological properties of microglia, including microglia-like morphology, expression of proper markers, and active phagocytosis. Additionally, documentation for isolating and labeling synaptosome substrates derived from human iPSC-derived lower motor neurons (i3LMNs) is provided. A live-cell, longitudinal imaging assay is used to monitor engulfment of human synaptosomes labeled with a pH-sensitive dye, allowing for investigations of iMG's phagocytic capacity. The protocols described herein are broadly applicable to different fields that are investigating human microglia biology and the contribution of microglia to disease.

Human Microglia-like Cells: Differentiation from Induced Pluripotent Stem Cells and In Vitro Live-cell Phagocytosis Assay using Human Synaptosomes

This protocol describes the differentiation process of human induced pluripotent stem cells (iPSCs) into microglia-like cells for in vitro experimentation. We also include a detailed procedure for generating human synaptosomes from iPSC-derived lower motor neurons that can be used as a substrate for in vitro phagocytosis assays using live-cell imaging systems.

Humane Mikroglia-ähnliche Zellen: Differenzierung aus induzierten pluripotenten Stammzellen und In-vitro-Lebendzell-Phagozytose-Assay mit humanen Synaptosomen

Microglia are the resident immune cells of myeloid origin that maintain homeostasis in the brain microenvironment and have become a key player in multiple ...