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.).

O consentimento informado do paciente conforme aprovado pelo conselho de revisão institucional deve ser obtido para o uso de células humanas. 1. Densidade de isolamento gradiente de sangue periférico humano as células mononucleares (PBMC) <ol><li> Adicionar 25 ml de sangue total heparinizado em um tubo de 50 ml por uma pipeta de 25 ml. <ol><li> Dilui-se as células com 25 ml de solução salina tamponada com fosfato (PBS). </li></ol></li><li> Adicionar 15 ml de gradiente de densidade d...

<ol>
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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...

Células estaminais mesenquimais humanas (MSCs) são progenitores somáticas que podem se diferenciar em linhagens mesodérmicos paraxiais as de osso, cartilagem, tecido adiposo e 1-4, bem como a um extramesodermal algumas linhagens 5. Primeiro isolada a partir da medula óssea adulta, estes progenitores multilinhagem foram agora encontrados em vários tecidos e 6-8, inesperadamente, mostraram ter fortes propriedades imunomoduladoras que aparecem altamente passível de aplicação clínica 9-12. Mecanismos detalhados envolvidos nos efeitos imunomoduladores estão ativamente sendo investigado por aplicação efectiva sobre as entidades de doenças específicas. Uma das maneiras mais simples para avaliar imunomodulação é através da avaliação para a supressão da efetoras de leucócitos proliferação 13. A maioria dos leucócitos, tais como linfócitos efectores T e monócitos proliferar prodigiously quando estimulado ou activado. Função imunomoduladora pode ser avaliado quando a supressão deproliferação é evidenciado. Tradicionalmente, a proliferação de leucócitos efectoras foi avaliada por detecção de [3 H] timidina em ADN. No entanto, este método tem desvantagens significativas, devido às preocupações de radiação e descarte pós-uso, bem como os equipamentos complexos necessários. Embora existam ensaios não radioactivos a fim de avaliar a proliferação celular, o éster de succinimidilo carboxifluoresceína (CFSE) ensaio tem outras vantagens tais como permitindo a identificação de populações celulares específicas, o que é especialmente útil em experiências de co-cultura, envolvendo vários tipos de células. CFSE é um corante fluorescente que celular pode ser avaliada por análise de citometria de fluxo. Como as células se dividem, a intensidade desta etiqueta celular é diminuída proporcionalmente; Isto não só possibilita a determinação da proliferação celular em geral, mas também permite a avaliação do número de divisões celulares até 8 divisões a fluorescência antes torna-se difícil para detect contra sinal de fundo. Além disso, a estabilidade do CFSE fluorescente in vivo permite a identificação de células que tais células marcadas podem ser visualizadas até diversos meses 14. Este ensaio pode também ser variada para avaliar tipos específicos de leucócitos efectoras ou a função imunomodulatória de populações específicas de MSC induzida por leucócitos, tais imunomoduladores como monócitos CD14 + a interleucina-10 (IL-10), produzindo 15 efectuando magnética talão selecção de marcadores de superfície populações de células de interesse antes ou após a co-cultura, conforme adequado. Nosso protocolo descreve o ensaio de base de avaliar os efeitos imunomoduladores de MSCs em leucócitos efectoras (diagrama de fluxo mostrado na Figura 1) e uma variação deste ensaio de base para a avaliação da induzida por MSC imunomodulação de leucócitos nos linfócitos CD4 + efectoras alogénicas T (fluxograma mostrado na Figura 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.

A Figura 1 indica o esquema geral do experimento, e A Figura 2 demonstra a aparência das várias condições de cultura de células tal como visualizado por microscopia de contraste de fase invertida. MSC são células aderentes com um fibroblástica, morfologia fusiforme, enquanto que PBMCs e leucócitos são pequenas células não-aderentes redondos. Estes dois tipos de células morfologicamente diferentes pode ser claramente visto na co-cultura. No fim do ensaio, quando os PBMC (ou leuckotyes) são aspi...

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

As propriedades imunomoduladoras de células-tronco mesenquimais humanas (MSC) aparecem cada vez mais relevante para a aplicação clínica. Usando um sistema de co-cultura de MSCs e leucócitos do sangue periférico de pré-corados com o éster succinimidilo corante fluorescente carboxifluoresceína (CFSE), descreve-se a avaliação in vitro de MSC imunomodulação em proliferação e subpopulações efectoras de leucócitos específicos.

Avaliação das propriedades imunomoduladora de mesenquimais humanas Células-Tronco (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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