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.).

Patient informerat samtycke som har godkänts av Institutional Review Board måste erhållas för användning av mänskliga celler. 1. densitetsgradient Isolering av humana perifera mononukleära blodceller (PBMC) <ol><li> Tillsätt 25 ml hepariniserade helblod i en 50 ml rör av en 25 ml pipett. <ol><li> Späd cellerna med 25 ml fosfatbuffrad saltlösning (PBS). </li></ol></li><li> Tillsätt 15 ml av Ficoll-Paque densitetsgradient i en ny 50 ml-rör, och medan luta röret, mycket långsam...

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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...

Humana mesenkyma stamceller (MSCs) är somatiska stamceller som kan differentieras till det paraxiella mesodermala linjerna av ben, brosk, och fettvävnad 1-4, såväl som till några extramesodermal härstamningar 5. Först isolerad från vuxen benmärg, har dessa multilineage förfäder nu finns i många vävnader 6-8 och oväntat visat sig ha starka immunmodulerande egenskaper som visas mycket mottagliga för klinisk tillämpning 9-12. Detaljerade mekanismer som är involverade i immunmodulerande effekter aktivt utreds för en effektiv tillämpning på specifika sjukdoms enheter. En av de mest enkla sätt att utvärdera immunmodulering är genom att bedöma för undertryckande av effektor leukocyt spridning 13. De flesta effektor leukocyter såsom T-lymfocyter och monocyter prolifererar svindlande när de stimuleras eller aktiveras. Immunmodulerande funktion kan bedömas vid undertryckande avspridning styrks. Traditionellt har effektor leukocyt proliferation utvärderats genom detektion av [3 H] tymidininkorporering i DNA. Emellertid har denna metod betydande nackdelar på grund av de problem som strålning och efter användning förfogande, liksom den komplexa utrustning som behövs. Även om det finns icke-radioaktiva analyser för att bedöma cellproliferation, har karboxifluorescein succinimidylester (CFSE) -analys andra fördelar såsom att låta för identifiering av specifika cellpopulationer, vilket är särskilt användbart i sam-kulturexperiment involverar flera celltyper. CFSE är en fluorescerande cell färgämne som kan bedömas genom flödescytometrisk analys. Som celler dela, är intensiteten i denna cellulära etikett minskas proportionellt; detta möjliggör inte bara bestämning av totala cellproliferation men tillåter också för bedömning av antalet celldelningar upp till 8 divisioner innan fluorescensen blir svårt att DETect mot bakgrundssignal. Vidare stabiliteten av det fluorescerande CFSE medger in vivo-spårning av märkta celler sådana att celler kan visualiseras upp till flera månader 14. Denna analys kan också varieras för att utvärdera specifika typer av effektorceller leukocyter eller den immunmodulatoriska funktionen av specifika populationer av MSC-inducerad immunmoduler leukocyter-såsom interleukin-10 (IL-10) som producerar CD14 + monocyter 15 genom att utföra magnetisk pärla ytmarkör urval av cellpopulationer av intresse före eller efter samodling på lämpligt sätt. Vår protokoll beskriver den grundläggande analysen enligt bedöma immunmodulerande effekter av MSC: er på effektor leukocyter (flödesschema som visas i fig 1) och en variant på denna grundläggande analys för utvärdering av MSC-inducerad leukocyt immunmodule på allogena CD4 + effektor-T-lymfocyter (flödesschema som visas i fig 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>
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		<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>
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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 betecknar den totala schemat av experimentet, och fig 2 visar utseendet hos de olika cellodlingsbetingelser som visualiseras genom faskontrast inverterad mikroskopi. MSC: er är vidhäftande celler med en fibroblastisk, spolformad morfologi, medan PBMC och leukocyter är små runda icke vidhäftande celler. Dessa två morfologiskt olika celltyper kan tydligt ses i co-kultur. I slutet av analysen, när PBMC (eller leuckotyes) aspireras för flödescytometrisk analyser, även om vidh?...

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

De immunmodulerande egenskaper mänskliga mesenkymala stamceller (MSC) visas allt viktigare för klinisk tillämpning. Med hjälp av en samodlingssystem av MSC och perifera blodleukocyter pre-färgades med fluorescerande färgämne karboxifluorescein succinimidylester (CFSE), beskriver vi bedömningen in vitro av MSC immunmodulering på effektor leukocyt spridning och särskilda subpopulationer.

Bedömning av immunmodulerande egenskaper av humana mesenkymala stamceller (MSC: er)

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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Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells (MSCs)

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