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 de Ficoll-Paque em um novo tubo de 50 ml, e, enquanto a inclinação do tubo, muito lentamente e cuidadosamente adicionar 25 ml da suspensão diluída de células ao longo do gradiente de densidade, de modo que não há mistura do sangue total com o gradiente de densidade, isto é., há perturbação da interface do gradiente de densidade de sangue. </li><li> Centrifugar a suspensão a partir do passo 1.2 a 400 x g durante 30 a 40 min num rotor de balde oscilante-de temperatura controlada sem travão a 20 ° C. Nota: Três camadas distintas deve ser aparente após centrifugação: the camada superior de plasma estar; a camada clara de fundo sendo o gradiente de densidade Ficoll-Paque; e uma camada celular fina, sendo o meio PBMC </li><li> Aspirar e desprezar o camada superior por sucção com uma pipeta de Pasteur, com o cuidado de não perturbar a camada de células mononucleares de interfase (isto é, linfócitos, monócitos e plaquetas). </li><li> Recolher cuidadosamente a camada de células mononucleares com uma pipeta de 10 mL para um novo tubo de centrífuga de 50 ml. </li><li> Encher o tubo com 20 ml de PBS, misturar bem, e centrifugar a 300 x g durante 10 min a 20 ° C. Remover o sobrenadante completamente após centrifugação. </li><li> Ressuspender o sedimento celular em 20 ml de PBS e centrifugar a 200 x g durante 10-15 minutos a 20 ° C. Remover o sobrenadante completamente após centrifugação. Nota: Esta etapa remove as plaquetas-que são indesejados-nos PBMC; este passo pode ser repetido para assegurar a remoção completa das plaquetas. </li><li> Se nenhuma seleção de populações específicas édesejado, sedimento de células ressuspender em leucócitos meio completo (10% soro fetal de bovino (FBS), 1% de L-glutamina, e 1% de penicilina / estreptomicina, em meio RPMI-1640) para cultura após a realização de uma contagem de células 16 para suspender a 1 ml de médio por 10 7 PBMC antes de prosseguir para a próxima etapa. </li></ol> 2. Rotulagem magnética de Leucócitos Populações (pule para o passo 4 se nenhuma seleção de populações específicas é Obrigatório) <ol><li> Centrifugar a suspensão de PBMC a 300 × g durante 10 min e o sobrenadante aspirado completamente. </li><li> Ressuspender o sedimento celular em 80 ul de PBS por 10 7 células totais. </li><li> Adicionar 20 uL de pérolas magnéticas especificados (isto é, CD14 esferas magnéticas para isolamento de monócitos CD4, esferas magnéticas para isolamento de linfócitos T) por 10 7 células totais. </li><li> Misturar bem e incubar durante 15 min a 2 a 8 ° C no frigorífico. </li><li> Adicionar 1-2 ml de PBS por 10 7 células para lavar e centrifugara 300 x g durante 10 min. </li><li> Aspirar o sobrenadante e ressuspender completamente até 8 10 células em 500 ul de PBS. </li></ol> 3. Seleção Magnética de subpopulações de leucócitos Nota: Uma variedade de separadores de esférulas magnéticas e colunas estão disponíveis para o isolamento de populações específicas de leucócitos pelo marcador de superfície celular de uma série de fabricantes. O isolamento pode ser por selecção positiva com base em um ou alguns marcadores positivos ou por selecção negativa após a depleção de populações indesejadas. Uma abordagem geral de efectuar a selecção positiva usando um marcador (isto é, CD4 de linfócitos T auxiliares, ou de monócitos CD14) é descrito a seguir. <ol><li> Preparar e separador magnético privilegiada incluindo coluna de separação de acordo com as instruções do fabricante. </li><li> Aplicar tubo contendo a amostra de PBMCs rotulados. Fornecer dois tubos de centrífuga de 15 ml para recolha do e unlabel marcado positivamentefrações celulares ed, seguindo as instruções do fabricante. </li><li> Contar o número de células 15 e ressuspender os leucócitos seleccionados positivamente (isto é, linfócitos T CD4 + ou CD14 + células) em meio completo de leucócitos, usando 1 ml de meio por 10 7 células. </li></ol> 4. CFSE Coloração de leucócitos para avaliação da proliferação Nota: CFSE tem sido amplamente utilizada em investigações imunológicas, tanto in vivo e in vitro. Nosso protocolo foi otimizado para estudo in vitro da MSC / PBMC (ou outros leucócitos) interações. Enquanto os passos gerais envolvidos na realização de CFSE marcação in vitro são semelhantes, pode haver diferenças na dose específica e tempo do protocolo 14. Isto pode ser devido a um número de factores, isto é, o tipo específico de célula utilizado, o número de células utilizadas-que podem provavelmente afectar a intensidade do sinal fluorescente CFSE. <ol&gt; <li> Dilui-se a solução de estoque de CFSE (10 mM) em PBS para a concentração de trabalho pretendida de 10 ^ M (solução de trabalho CFSE-). Prepare 10 7 células (isto é, PBMCs ou células T) para marcação com a solução CFSE-working. </li><li> Centrifuga-se a 300 x g durante 10 minutos para se obter uma pelete de células e o sobrenadante aspirado. </li><li> Ressuspender as células suavemente em 1 ml de solução de pré-aquecido (37 ° C) CFSE-trabalho e incubar as células durante 10 min a 37 ° C. </li><li> Para lavar o excesso de CFSE, diluir a suspensão de células com 10 vezes (em volume) de pré-arrefecida (4 ° C), meio RPMI contendo 10% de FBS. Sedimentar as células por centrifugação a 300 xg durante 5 min e desprezar o sobrenadante. Lava-se a pelete de células desta forma duas vezes mais. </li><li> Re-sedimentar as células por centrifugação e contar o número de células 15. Ressuspender 10 7 células (quer PBMCs ou células T) em 1 mL de meio completo pré-aquecido de leucócitos fresco. </li></ol> 5. Co-cultura de MSCs com leucócitos e activação de leucócitos <ol><li> Pré-quente MSC meio completo (10% de FBS (pré-testados para o crescimento óptimo MSC), 1% de L-glutamina, e 1% de penicilina / estreptomicina, em meio DMEM-baixo teor de glucose) a 37 ° C durante não mais do que 30 min. </li><li> MSCs semente a 50.000 células em 1 ml de MSC meio completo em placas de 24 poços (densidade MSC: 25.000 células / cm2) para a ligação O / N em uma temperatura de 37 ° C incubadora, permitindo para as células estaminais para alcançar 80% de confluência. </li><li> Meio aspirado, e com base em números de MSC sem sementes, adicionar PBMC CFSE-marcadas (previamente marcado com consulte protocolo Passo 4 acima) para as MSCs cultivadas (semeadas no dia anterior em placas de 24 cavidades) em 1 ml de leucócitos meio completo a uma 01:10 (índice de célula) rácio co-cultura de MSCs para PBMC. </li><li> Adicionar o mitogénio fito-hemaglutinina (PHA), um activador de leucócitos não específica, a uma concentração final de 10 ug / ml em um volume total de 1 ml de leucócitos meio completo por poço. <ol><li> AlternativEly, para estimular a activação de linfócitos T especificamente: a-usar CD3 / 28 micropérolas e adicionar à co-cultura de duas células para obter uma razão de 1 talão de linfócitos-T-a: 1. </li></ol></li><li> Para controlo negativo, placa 500.000 PBMCs / poço (ou uma população efectora específica de leucócitos; densidade celular: 250.000 leucócitos / cm2) numa placa de 24 cavidades com 1 ml de meio completo de leucócitos única; para o controlo positivo, para além do plaqueamento o mesmo número de PBMC / cavidade, adicionar PHA a uma concentração final de 10 ug / ml. </li><li> No terceiro e quinto dia da experiência de co-cultura, avaliar a proliferação de leucócitos marcados com CFSE (colocados em tubos de fundo redondo) por análise de citometria de fluxo 17 com 488 nm de excitação e emissão adequados para filtros de fluoresceína. Nota: Coloração de citocinas intracelulares para a análise de citometria de fluxo pode também ser realizada para estes leucócitos marcados com CFSE, neste ponto, para avaliar alterações no perfil de expressão de citoquinas de leucócitos como MOdulated por MSCs. Desde CFSE é avaliada com um filtro adequado para a fluoresceína, os anticorpos seleccionados para avaliar várias citocinas precisa ser conjugado com fluoresceína ou com excepção espectro semelhante fluorocromos (isto é, ficoeritrina, proteína peridinina clorofila complexo (PerCP)). </li></ol> 6. Variação: Effector Suppression Ensaio Magnetic Bead-selecionado, MSC-induzida imunomoduladora leucócitos ativados em CFSE rotulados células efetoras T CD4 + <ol><li> MSCs semente a 250.000 células em 3 ml de MSC meio completo em placas de 6 poços (densidade MSC: 25.000 células / cm2) para a ligação O / N em uma temperatura de 37 ° C incubadora, permitindo para as células estaminais para alcançar 80% de confluência. Pelo menos 3 placas de 6 poços são necessárias para garantir PBMC MSC-cocultivadas suficientes para a seleção sub-população. </li><li> Aspirar o meio e adicionar PBMC separados de acordo com Passo 1, mas em placas de 6 poços, a 2,5 x 10 6 células / poço (densidade celular: 250.000 leukocytes / cm 2) com 3 ml de meio completo de leucócitos. A co-cultura durante 48-72 horas numa incubadora a 37 ° C. </li><li> Magnetic talão-selecionar população específica de leucócitos induzidos imunomoduladores-MSC (ou seja, CD14 + células) como por secções 2-3. Notas: a coloração de citocinas intracelulares para a análise de citometria de fluxo pode ser realizada neste ponto para avaliar mudanças no perfil de expressão de citoquinas de leucócitos, isto é, a expressão de interleucina-10 como modulada por MSC. </li><li> Adicionar CFSE marcado com células T CD4 + alogeneicas gerados como por secções 2-4 em placas de 24 poços em 1 ml de leucócitos completo (densidade de células T: 250.000 células / cm 2) forma a leucócitos induzida por MSC seleccionado por esferas em várias proporções , ou seja, 1:10, 1: 5, 1: 2 e 1: 1 (célula para célula) as proporções. </li><li> Para estimular as células T CD4 +, adicionar cc-CD3 / 28 micropérolas conjugados para obter uma razão talão-a-célula de 1: 1. </li><li> Para um controlo negativo, a chapa 500.000 células T CD4 + / poço num (a densidade de células T: 250000 células / cm 2) de 24 poços da placa com 1 ml de meio completo de leucócitos única; para o controlo positivo, para além do plaqueamento o mesmo número de células T CD4 + / cavidade, adicionar a-CD3 / 28 micropérolas conjugados para obter uma razão talão-a-célula de 1: 1. </li><li> No dia 3 de co-cultura de, avaliar a proliferação de células T CD4 + CFSE-marcados (colocados em tubos de fundo redondo) por análise citométrica de fluxo 16 com 488 nm de excitação e emissão adequados para filtros de fluoresceína. </li></ol>

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We have nothing to disclose.

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).

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			<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 ...

assessment-immunomodulatory-properties-human-mesenchymal-stem-cells

Research

JoVE Journal

Immunology and Infection

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

12.1K visualizações.  National Health Research Institutes (NHRI).  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. 

Vídeo: Assessment of the Immunomodulatory Properties of Human Mesenchymal Stem Cells MSCs

Expansion, Purification, and Functional Assessment of Human Peripheral Blood NK Cells

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of NK cells and ability to expand in vitro has restricted development of NK cell immunotherapy. Here we describe a method to efficiently expand vast quantities of functional NK cells ex vivo using K562 cells expressing membrane-bound IL21, as an artificial antigen-presenting cell (aAPC).
NK cell adoptive therapies to date have utilized a cell product obtained by steady-state leukapheresis of the donor followed by depletion of T cells or positive selection of NK cells. The product is usually activated in IL-2 overnight and then administered the following day 1. Because of the low frequency of NK cells in peripheral blood, relatively small numbers of NK cells have been delivered in clinical trials.
The inability to propagate NK cells in vitro has been the limiting factor for generating sufficient cell numbers for optimal clinical outcome. Some expansion of NK cells (5-10 fold over 1-2 weeks) has be achieved through high-dose IL-2 alone 2. Activation of autologous T cells can mediate NK cell expansion, presumably also through release of local cytokine 3. Support with mesenchymal stroma or artificial antigen presenting cells (aAPCs) can support the expansion of NK cells from both peripheral blood and cord blood 4. Combined NKp46 and CD2 activation by antibody-coated beads is currently marketed for NK cell expansion (Miltenyi Biotec, Auburn CA), resulting in approximately 100-fold expansion in 21 days.
Clinical trials using aAPC-expanded or -activated NK cells are underway, one using leukemic cell line CTV-1 to prime and activate NK cells5 without significant expansion. A second trial utilizes EBV-LCL for NK cell expansion, achieving a mean 490-fold expansion in 21 days6. The third utilizes a K562-based aAPC transduced with 4-1BBL (CD137L) and membrane-bound IL-15 (mIL-15)7, which achieved a mean NK expansion 277-fold in 21 days. Although, the NK cells expanded using K562-41BBL-mIL15 aAPC are highly cytotoxic in vitro and in vivo compared to unexpanded NK cells, and participate in ADCC, their proliferation is limited by senescence attributed to telomere shortening8. More recently a 350-fold expansion of NK cells was reported using K562 expressing MICA, 4-1BBL and IL159.
Our method of NK cell expansion described herein produces rapid proliferation of NK cells without senescence achieving a median 21,000-fold expansion in 21 days.

Here we describe a method to efficiently expand and purify large numbers of human NK cells and assess their function.

Expansão, Purificação e Avaliação Funcional de Células NK do sangue periférico

Natural killer (NK) cells play an important role in immune surveillance against a variety of infectious microorganisms and tumors. Limited availability of ...

Imunologia e Infecção

Quantitative Assessment of Immune Cells in the Injured Spinal Cord Tissue by Flow Cytometry:  a Novel Use for a Cell Purification Method

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true quantitative analysis of cellular inflammation. Flow cytometry is a quick alternative method to quantify immune cells in the injured brain or spinal cord tissue. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue. However, the dissociated spinal cord tissue is concentrated with myelin debris that can be mistaken for cells and reduce cell count reliability obtained by the flow cytometer. We have advanced a cell preparation method using the OptiPrep gradient system to effectively separate lipid/myelin debris from cells, providing sensitive and reliable quantifications of cellular inflammation in the injured spinal cord by flow cytometry. As described in our recent study (Beck &amp; Nguyen et al., Brain. 2010 Feb; 133 (Pt 2): 433-47), the OptiPrep cell preparation had increased sensitivity to detect cellular inflammation in the injured spinal cord, with counts of specific cell types correlating with injury severity. Critically, novel usage of this method provided the first characterization of acute and chronic cellular inflammation after SCI to include a complete time course for polymorphonuclear leukocytes (PMNs, neutrophils), macrophages/microglia, and T-cells over a period ranging from 2 hours to 180 days post-injury (dpi), identifying a surprising novel second phase of cellular inflammation. Thorough characterization of cellular inflammation using this method may provide a better understanding of neuroinflammation in the injured CNS, and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for SCI.

Quantification of cellular inflammation in the injured/pathological CNS by flow cytometry is complicated by lipid/myelin debris that can have similar size and granulation to cells, decreasing sensitivity/accuracy. We have advanced a cell preparation method to remove myelin debris and improve cell detection by flow cytometry in the injured spinal cord.

Avaliação quantitativa das células imunes no tecido medula lesionada por Citometria de Fluxo: um novo uso para um método de purificação celular

Detection of immune cells in the injured central nervous system (CNS) using morphological or histological techniques has not always provided true ...

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, transmission of antibiotic resistant bacteria between animals and humans, and the high cost of treating infections.
In this study, we disclose a new strategy that may be effective in preventing implant-associated infection based on the potential antimicrobial properties of platelet-rich plasma (PRP). Due to its well-studied properties for promoting healing, PRP (a biological product) has been increasingly used for clinical applications including orthopaedic surgeries, periodontal and oral surgeries, maxillofacial surgeries, plastic surgeries, sports medicine, etc.
PRP could be an advanced alternative to conventional antibiotic treatments in preventing implant-associated infections. The use of PRP may be advantageous compared to conventional antibiotic treatments since PRP is less likely to induce antibiotic resistance and PRP's antimicrobial and healing-promoting properties may have a synergistic effect on infection prevention. It is well known that pathogens and human cells are racing for implant surfaces, and PRP's properties of promoting healing could improve human cell attachment thereby reducing the odds for infection. In addition, PRP is inherently biocompatible, and safe and free from the risk of transmissible diseases.
For our study, we have selected several clinical bacterial strains that are commonly found in orthopaedic infections and examined whether PRP has in vitro antimicrobial properties against these bacteria. We have prepared PRP using a twice centrifugation approach which allows the same platelet concentration to be obtained for all samples. We have achieved consistent antimicrobial findings and found that PRP has strong in vitro antimicrobial properties against bacteria like methicillin-sensitive and methicillin-resistant Staphylococcus aureus, Group A Streptococcus, and Neisseria gonorrhoeae. Therefore, the use of PRP may have the potential to prevent infection and to reduce the need for costly post-operative treatment of implant-associated infections.

PRP as a New Approach to Prevent Infection: Preparation and In vitro Antimicrobial Properties of PRP

Implant-associated infection is a significant clinical complication. This study describes an approach using platelet-rich plasma (PRP) to prevent implant-associated infections, presents the protocol for preparing PRP with constant platelet concentration, and reports the newly identified antimicrobial properties of PRP and related protocols for examining such antimicrobial properties in vitro.

PRP como uma nova abordagem para prevenir a infecção: Preparação e<em&gt; In vitro</em&gt; Propriedades antimicrobianas do PRP

Implant-associated infection is becoming more and more challenging to the healthcare industry worldwide due to increasing antibiotic resistance, ...

Development of Stem Cell-derived Antigen-specific Regulatory T Cells Against Autoimmunity

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic self-tolerance. Tregs represent about 5 - 10% of the mature CD4+ T cell subpopulation in mice and humans, with about 1 - 2% of those Tregs circulating in the peripheral blood. Induced pluripotent stem cells (iPSCs) can be differentiated into functional Tregs, which have a potential to be used for cell-based therapies of autoimmune diseases. Here, we present a method to develop antigen (Ag)-specific Tregs from iPSCs (i.e., iPSC-Tregs). The method is based on incorporating the transcription factor FoxP3 and an Ag-specific T cell receptor (TCR) into iPSCs and then differentiating on OP9 stromal cells expressing Notch ligands delta-like (DL) 1 and DL4. Following in vitro differentiation, the iPSC-Tregs express CD4, CD8, CD3, CD25, FoxP3, and Ag-specific TCR and are able to respond to Ag stimulation. This method has been successfully applied to cell-based therapy of autoimmune arthritis in a murine model. Adoptive transfer of these Ag-specific iPSC-Tregs into Ag-induced arthritis (AIA)-bearing mice has the ability to reduce joint inflammation and swelling and to prevent bone loss.

We present here a method to develop functional antigen (Ag)-specific regulatory T cells (Tregs) from induced pluripotent stem cells (iPSCs) for immunotherapy of autoimmune arthritis in a murine model.

Desenvolvimento de Células-Tronco de derivados de células T regulatórias antígeno específico da Contra Auto-imunidade

Autoimmune diseases arise due to the loss of immunological self-tolerance. Regulatory T cells (Tregs) are important mediators of immunologic ...

Dextran Enhances the Lentiviral Transduction Efficiency of Murine and Human Primary NK Cells

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining the role of the gene of interest in the development, differentiation, and function of NK cells. Recent advances related to chimeric antigen receptors (CARs) in cancer immunotherapy accentuate the need for an efficient method to deliver exogenous genes to effector lymphocytes. The efficiencies of lentiviral-mediated gene transductions into primary human or mouse NK cells remain significantly low, which is a major limiting factor. Recent advances using cationic polymers, such as polybrene, show an improved gene transduction efficiency in T cells. However, these products failed to improve the transduction efficiencies of NK cells. This work shows that dextran, a branched glucan polysaccharide, significantly improves the transduction efficiency of human and mouse primary NK cells. This highly reproducible transduction methodology provides a competent tool for transducing human primary NK cells, which can vastly improve clinical gene delivery applications and thus NK cell-based cancer immunotherapy.

The goal of this study was to formulate technologies that allow for successful gene transduction in primary natural killer (NK) cells. The dextran-mediated lentiviral transduction of human or mouse primary NK cells results in higher gene expression efficiencies. This method of gene transduction will vastly improve NK cell genetic manipulation.

Dextran aumenta a eficiência de Lentivirus transdução das células NK primário murino e humano

The efficient transduction of specific genes into natural killer (NK) cells has been a major challenge. Successful transductions are critical to defining ...

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode larvae of the genus Trichinella. The most important sources of human infections worldwide are game meat and pork or pork products. In many countries, the prevention of human trichinellosis is based on the identification of infected animals by means of the artificial digestion of muscle samples from susceptible animal carcasses.
There are several methods based on the digestion of meat but the magnetic stirrer method is considered the gold standard. This method allows the detection of Trichinella larvae by microscopy after the enzymatic digestion of muscle samples and subsequent filtration and sedimentation steps. Although this method does not require special and expensive equipment, internal controls cannot be used. Therefore, stringent quality management should be applied throughout the test. The aim of the present work is to provide detailed handling instructions and critical control points of the method to analysts, based on the experience of the European Union Reference Laboratory for Parasites and the National Reference Laboratory of Germany for Trichinella.

Magnetic Stirrer Method for the Detection of Trichinella Larvae in Muscle Samples

The prevention of human trichinellosis in many countries worldwide is based on the laboratory examination of muscle samples from susceptible animals by methods of digestion, of which the magnetic stirrer method is considered the gold standard.

Método agitador magnético para Detecção de<em&gt; Trichinella</em&gt; As larvas em amostras de músculo

Trichinellosis is a debilitating disease in humans and is caused by the consumption of raw or undercooked meat of animals infected with the nematode ...

A Flow Cytometry-Based Cytotoxicity Assay for the Assessment of Human NK Cell Activity

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, specifically eliminating tumoral and virally infected cells. Approximately 30 known monogenic defects, together with a host of other pathological conditions, cause either functional or classic NK cell deficiency, manifesting in reduced or absent cytotoxic activity. Historically, cytotoxicity has been investigated with radioactive methods, which are cumbersome, expensive and potentially hazardous. This article describes a streamlined, clinically applicable flow cytometry-based method to quantify NK cell cytotoxic activity. In this assay, peripheral blood mononuclear cells (PBMCs) or purified NK cell preparations are co-incubated at different ratios with a target tumor cell line known to be sensitive to NK cell-mediated cytotoxicity (NKCC). The target cells are pre-labeled with a fluorescent dye to allow their discrimination from the effector cells (NK cells). After the incubation period, killed target cells are identified by a nucleic acid stain, which specifically permeates dead cells. This method is amenable to both diagnostic and research applications and, thanks to the multi-parameter capabilities of flow cytometry, has the added advantage of potentially enabling a deeper analysis of NK cell phenotype and function.

A flow cytometry-based method to quantitatively determine the cytotoxic activity of human natural killer cells is shown here.

Um ensaio de citotoxicidade baseados em citometria de fluxo para a avaliação da atividade das células NK humana

Within the innate immune system, effector lymphocytes known as natural killer (NK) cells play an essential role in host defense against aberrant cells, ...

Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new therapeutics. Additionally, registration of new substances requires appropriate risk assessment with adequate testing systems to avoid the risk of individuals being harmed, for example, in the working environment. Such risk assessments are usually conducted in animal studies. In view of the 3Rs principle and public skepticism against animal experiments, human alternative methods, such as precision-cut lung slices (PCLS), have been evolving. The present paper describes the ex vivo technique of human PCLS to study the immunomodulatory potential of low-molecular-weight substances, such as ammonium hexachloroplatinate (HClPt). Measured endpoints include viability and local respiratory inflammation, marked by altered secretion of cytokines and chemokines. Pro-inflammatory cytokines, tumor necrosis factor alpha (TNF-&#945;), and interleukin 1 alpha (IL-1&#945;) were significantly increased in human PCLS after exposure to a sub-toxic concentration of HClPt. Even though the technique of PCLS has been substantially optimized over the past decades, its applicability for the testing of immunomodulation is still in development. Therefore, the results presented here are preliminary, even though they show the potential of human PCLS as a valuable tool in respiratory research.

In view of the 3Rs principle, respiratory models as alternatives to animal studies are evolving. Especially for risk assessment of respiratory substances, there is a lack of appropriate assays. Here, we describe the use of human precision-cut lung slices for the assessment of airborne substances.

Avaliação do citotóxicos e efeitos imunomoduladores de substâncias no pulmão humano de precisão de corte fatias

Respiratory diseases in their broad diversity need appropriate model systems to understand the underlying mechanisms and enable development of new ...

Assessment of the Synaptic Interface of Primary Human T Cells from Peripheral Blood and Lymphoid Tissue

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of glass-supported planar bilayers and in vitro-derived T-cell clones or lines1,2,3,4. How these findings apply to the primary human T cells isolated from blood or lymphoid tissues is not known, partly due to significant difficulties in obtaining a sufficient number of cells for analysis5. Here we address this through the development of a technique exploiting multichannel flow slides to build planar lipid bilayers containing activating and adhesion molecules. The low height of the flow slides promotes rapid cell sedimentation in order to synchronize cell:bilayer attachment, thereby allowing researchers to study the dynamic of the synaptic interface formation and the kinetics of the granules release. We apply this approach to analyze the synaptic interface of as few as 104 to 105 primary cryopreserved T cells isolated from lymph nodes (LN) and peripheral blood (PB). The results reveal that the novel planar lipid bilayer technique enables the study of the biophysical properties of primary human T cells derived from blood and tissues in the context of health and disease.

The protocol describes a technique to study the ability of primary polyclonal human T cells to form synaptic interfaces using planar lipid bilayers. We use this technique to show the differential synapse formation capability of human primary T cells derived from lymph nodes and peripheral blood.

Avaliação da Interface sináptica de células T humano primário do sangue periférico e tecido linfoide

The current understanding of the dynamics and structural features of T-cell synaptic interfaces has been largely determined through the use of ...

Induced Differentiation of M Cell-like Cells in Human Stem Cell-derived Ileal Enteroid Monolayers

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where immune cells reside and therefore contribute to mucosal immunity in the intestine. A complete understanding of how M cells differentiate in the intestine as well as the molecular mechanisms of antigen uptake by M cells is lacking. This is because M cells are a rare population of cells in the intestine and because in vitro models for M cells are not robust. The discovery of a self-renewing stem cell culture system of the intestine, termed enteroids, has provided new possibilities for culturing M cells. Enteroids are advantageous over standard cultured cell lines because they can be differentiated into several major cell types found in the intestine, including goblet cells, Paneth cells, enteroendocrine cells and enterocytes. The cytokine RANKL is essential in M cell development, and addition of RANKL and TNF-&#945; to culture media promotes a subset of cells from ileal enteroids to differentiate into M cells. The following protocol describes a method for the differentiation of M cells in a transwell epithelial polarized monolayer system of the intestine using human ileal enteroids. This method can be applied to the study of M cell development and function.

This protocol describes how to induce the differentiation of M cells in human stem cell-derived ileal monolayers and methods to assess their development.

Diferenciação induzida de pilhas de M cell-like em monolayers Enteroid ileal derivado da pilha de haste humana

M (microfold) cells of the intestine function to transport antigen from the apical lumen to the underlying Peyer&#8217;s patches and lamina propria where ...