Name: assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)
Uploaded: 2015-12-24T14:00:00
Description: Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

This work was supported in part by grants from NHRI (CS-104-PP-06 to B.L.Y.).

Patientenzustimmung, wie durch die Ethikkommission genehmigt muss für die Verwendung von menschlichen Zellen gewonnen werden. 1. Dichtegradienten Isolierung von humanen peripheren mononukleären Blutzellen (PBMCs) <ol><li> 25 ml heparinisiertes Vollblut in eine 50-ml-Röhrchen mit einer 25 ml Pipette. <ol><li> Zellen verdünnt mit 25 ml phosphatgepufferter Salzlösung (PBS). </li></ol></li><li> 15 ml Ficoll-Paque-Dichtegradienten in ein neues 50 ml Röhre und beim Kippen der Röhre, sehr ...

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Increasingly, the immunomodulatory properties of MSCs are being translated into clinical use more rapidly than the multilineage capacity of these stem cells18-20. Thus, co-culture techniques of MSCs with leukocytes and assays to evaluate immune function are important to further delineate the specific mechanisms involved in these properties for optimizing effective therapeutic application. 
One of the most critical technical aspects for success in these assays is having adequate PBMC...

Humane mesenchymale Stammzellen (MSCs) sind somatische Vorläufern, die in die achsparallel mesodermalen Abstammungslinien von Knochen, Knorpel und Fettgewebe 1-4, um ein paar extramesodermal Linien 5 unterscheiden kann, wie auch. Zuerst von der erwachsenen Knochenmark isoliert wurden diese multilineage Vorläufern jetzt in zahlreichen Geweben 6-8 gefunden und unerwarteterweise gezeigt, dass starke immunmodulatorische Eigenschaften, die sehr gut für die klinische Anwendung 9-12 erscheinen müssen. Detaillierte Mechanismen in den immunmodulatorische Effekte einbezogen werden aktiv für eine wirksame Anwendung auf spezifische Krankheitsentitäten untersucht. Eine der direkten Weg zur Immunmodulation zu bewerten, ist durch die Ermittlung für die Unterdrückung der Proliferation von Leukozyten-Effektor 13. Effektor meisten Leukozyten wie T-Lymphozyten und Monozyten zu vermehren prodigiously wenn sie stimuliert oder aktiviert. Immunmodulatorische Funktion kann geprüft werden, wenn UnterdrückungProliferation wird belegt. Traditionell wurde Effektor Leukozyten Proliferation durch Detektion von [3 H] Thymidin-Einbau in die DNA untersucht. , Dieses Verfahren hat jedoch erhebliche Nachteile auf Grund der Bedenken von Strahlung und post-Nutzung zur Verfügung, ebenso wie die komplexe Ausrüstung erforderlich. Zwar gibt es nicht-radioaktiven Assays, die Zellproliferation zu beurteilen, hat das Carboxyfluorescein Succinimidylester (CFSE) -Assay andere Vorteile, wie beispielsweise, die eine Identifizierung von spezifischen Zellpopulationen, die besonders nützlich bei der Co-Kultur-Experimente mit mehreren Zelltypen. CFSE ein fluoreszierender Farbstoff, der zellulären durch durchflusszytometrische Analyse bestimmt werden kann. Wie Zellen sich teilen, wird die Intensität dieses zellulären Etikett proportional verringert; Dies ermöglicht nicht nur die Bestimmung der Gesamt-Zellproliferation, sondern ermöglicht auch die Beurteilung von der Anzahl der Zellteilungen bis 8 Einheiten, bevor die Fluoreszenz wird schwierig, detect gegen Hintergrundsignal. Darüber hinaus ist die Stabilität des fluoreszierenden CFSE ermöglicht in vivo Verfolgung von markierten Zellen, so daß Zellen können bis zu vielen Monaten 14 visualisiert werden. Dieser Assay kann auch variiert werden, um spezifische Typen von Effektorzellen Leukozyten oder immunmodulatorischen Funktion spezifischer Populationen auszuwerten MSC-induzierte immunmodulatorische Leukozyten-wie Interleukin-10 (IL-10) produzieren CD14 + Monocyten 15 durch Ausführen einer Magnetperle Oberflächenmarker Auswahl Zellpopulationen von Interesse vor oder nach der Ko-Kultur, wie angemessen. Unser Protokoll beschreibt die grundlegende Assay zur Beurteilung der immunmodulatorischen Wirkung von MSCs auf Effektorzellen Leukozyten (Flussdiagramm in Figur 1 gezeigt) und einer Variation dieser Basistest zur Bewertung der MSC-induzierte Leukozyten-Immunmodulation zur allogenen CD4 + Effektor-T-Lymphozyten (Flußdiagramm gezeigten in Abbildung 4).

<table border="0" cellpadding="0" cellspacing="0" width="1205">
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Ficoll-Paque PLUS</td>
			<td style="width:259px;">GE Healthcare</td>
			<td style="width:121px;">71-7167-00 AG</td>
			<td style="width:549px;">Density grandient for isolation of peripheral blood mononuclear cells (PBMCs)</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Vibrant CFDA-SE Cell Tracer Kit (CFSE)</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">V12883</td>
			<td>Cellular label for detection of cell division</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Phytoagglutinin (PHA)</td>
			<td style="width:259px;">Sigma</td>
			<td>L8902</td>
			<td>Activation of human PBMCs</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:276px;">Dynabeads Human T-Activator CD3/28</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">111.32D</td>
			<td style="width:549px;">Activation of human T lymphocytes, e.g. CD4+ T cells, CD8+ T cells,etc.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">autoMACS&#8482; Separator</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">autoMACS&#8482; Separator</td>
			<td>Magnetic based cell separator</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">autoMACS&#174; Columns</td>
			<td>Miltenyi Biotec</td>
			<td>130-021-101</td>
			<td>separation columns</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:276px;">CD14 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td>130-050-201</td>
			<td style="width:549px;">For positive selection of CD14+ human monocytes and macrophages from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">CD4 microbeads, human&#160;</td>
			<td>Miltenyi Biotec</td>
			<td style="width:121px;">130-045-101</td>
			<td style="width:549px;">For positive selection of CD4+ human T lymphocytes from PBMCs</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">RPMI 1640 Medium</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11875</td>
			<td>Human PBMC/leukocyte culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">DMEM, Low glucose, pyruvate</td>
			<td style="width:259px;">Life Technologies</td>
			<td style="width:121px;">11885</td>
			<td style="width:549px;">Human mesenchymal stem cell (MSC) culture medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">L-glutamine</td>
			<td style="width:259px;">Life Technologies</td>
			<td>25030-081</td>
			<td>Supplementation for MSC complete medium</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">Penicillin/Streptomycin</td>
			<td style="width:259px;">Life Technologies</td>
			<td>15070-063</td>
			<td>Supplementation for PBMC/leukocyte and MSC complete medium</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:276px;">Fetal bovine serum (FBS)</td>
			<td style="width:259px;">1) Hyclone, for MSC culture&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160;&#160; 2) Life Technologies, for all other cells (i.e. PBMCs, specific leukocyte populations)</td>
			<td style="width:121px;">1) SH30070.03M 2) 10091-148</td>
			<td>Pre-test lots for support of MSC in vitro culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">24-well cell culture plate</td>
			<td style="width:259px;">Corning</td>
			<td>COR3524</td>
			<td>Co-culture plate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">50 mL centrifuge tube</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430291</td>
			<td>Isolation PBMCs from whole blood by Ficoll-Paque PLUS</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:276px;">15 mL centrifuge tube&#160;</td>
			<td style="width:259px;">Corning</td>
			<td style="width:121px;">430766</td>
			<td>Collection of the labeled and unlabeled cell fractions</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Round-bottom tubes</td>
			<td style="width:259px;">BD Falcon&#160;</td>
			<td style="width:121px;">352008</td>
			<td>Collection of cells for flow cytometric analysis</td>
		</tr>
	</tbody>
</table>

assessment of the immunomodulatory properties of human mesenchymal stem cells (mscs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of immune-related diseases. Mechanisms involved are increasingly being elucidated and in this article, we describe the basic experiment to assess MSC immunomodulation by assaying for suppression of effector leukocyte proliferation. Representing activation, leukocyte proliferation can be assessed by a number of techniques, and we describe in this protocol the use of the fluorescent cellular dye carboxyfluorescein succinimidyl ester (CFSE) to label leukocytes with subsequent flow cytometric analyses. This technique can not only assess proliferation without radioactivity, but also the number of cell divisions that have occurred as well as allowing for identification of the specific population of proliferating cells and intracellular cytokine/factor expression. Moreover, the assay can be tailored to evaluate specific populations of effector leukocytes by magnetic bead surface marker selection of single peripheral blood mononuclear cell populations prior to co-culture with MSCs. The flexibility of this co-culture assay is useful for investigating cellular interactions between MSCs and leukocytes.

Figur 1 zeigt die Gesamtschema des Experiments, und Figur 2 zeigt das Aussehen der verschiedenen Zellkulturbedingungen, wie durch Phasenkontrastmikroskopie sichtbar invertiert. MSCs sind adhärente Zellen mit Fibroblasten, spindelförmige Morphologie, wobei PBMCs und Leukozyten sind kleine, runde, nichthaftende Zellen. Diese zwei morphologisch unterschiedliche Zelltypen ist deutlich in der Co-Kultur gesehen werden. Am Ende des Tests, wenn die PBMCs (oder leuckotyes) für durchflusszytometrische abgesaugt an...

Watch this Scientific Journal Video about Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs at JoVE.com

Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Die immunmodulatorischen Eigenschaften von humanen mesenchymalen Stammzellen (MSC) erscheint für die klinische Anwendung zunehmend relevant. Verwendung eines Co-Kultursystem der MSCs und peripheren Blutleukozyten mit dem Fluoreszenzfarbstoff Carboxyfluorescein Succinimidylester (CFSE) vorgefärbten beschreiben wir den in vitro Beurteilung der MSC Immunomodulation auf Effektorzellen Leukozyten Proliferation und bestimmten Subpopulationen.

Beurteilung der immunmodulatorischen Eigenschaften von humanen mesenchymalen Stammzellen (MSCs)

The immunomodulatory properties of multilineage human mesenchymal stem cells (MSCs) appear to be highly relevant for clinical use towards a wide-range of ...

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