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

El consentimiento informado del paciente aprobado por la junta de revisión institucional debe ser obtenido por el uso de las células humanas. 1. Densidad aislamiento degradado de células mononucleares de sangre periférica humana (PBMC) <ol><li> Añadir 25 ml de sangre entera heparinizada en un tubo de 50 ml con una pipeta 25 ml. <ol><li> Diluir las células con 25 ml de solución salina tamponada con fosfato (PBS). </li></ol></li><li> Añadir 15 ml de gradiente de densidad de Ficoll-Paque en un nuevo tubo de 50 ml, y mientras se inclina el tubo, muy lentamente y cuidadosamente añadir en 25 ml de la suspensión celular diluido a lo largo del gradiente de densidad de manera que no hay mezcla de la sangre entera con el gradiente de densidad, es decir., ninguna perturbación de la interfaz de gradiente de densidad de sangre. </li><li> Centrifugar la suspensión de la etapa 1.2 a 400 × g durante 30 a 40 min en un rotor oscilante-cubo de temperatura controlada sin freno a 20 ° C. Nota: Tres capas distintas deben ser evidentes después de la centrifugación: THe plasma ser capa superior; la capa transparente de fondo es el gradiente de densidad de Ficoll-Paque; y una capa celular fina, medio estando las PBMCs </li><li> Aspirar y descartar la capa superior por aspiración con una pipeta Pasteur, con cuidado de no alterar la capa de interfase de células mononucleares (es decir, linfocitos, monocitos y plaquetas). </li><li> Recoger cuidadosamente esta capa de células mononucleares con una pipeta de 10 ml a un nuevo tubo de centrífuga de 50 ml. </li><li> Llenar el tubo con 20 ml de PBS, se mezcla bien, y se centrifuga a 300 xg durante 10 min a 20 ° C. Eliminar el sobrenadante por completo después de la centrifugación. </li><li> Resuspender el sedimento celular en 20 ml de PBS y centrifugar a 200 × g durante 10-15 minutos a 20 ° C. Eliminar el sobrenadante por completo después de la centrifugación. Nota: Este paso elimina las plaquetas-que no son deseados, dentro de los PBMCs; este paso se puede repetir para asegurar la eliminación completa de las plaquetas. </li><li> Si no hay selección de poblaciones específicas esdeseada, sedimento celular se resuspende en medio completo de leucocitos (suero bovino fetal al 10% (FBS), 1% de L-glutamina, y 1% de penicilina / estreptomicina en medio RPMI-1640) para el cultivo después de realizar un recuento de células 16 para suspender 1 ml de medio por cada 10 7 PBMC antes de proceder al siguiente paso. </li></ol> 2. Etiquetado magnética de leucocitos Poblaciones (vaya al paso 4 si no hay selección de poblaciones específicas es necesario) <ol><li> Centrifugar la suspensión de PBMC a 300 × g durante 10 min y el sobrenadante aspirado por completo. </li><li> Resuspender el sedimento celular en 80 l de PBS por 10 7 células totales. </li><li> Añadir 20 l de perlas magnéticas especificados (es decir, perlas magnéticas CD14 para el aislamiento de monocitos, perlas magnéticas CD4 para el aislamiento de linfocitos T) por 10 7 células totales. </li><li> Mezclar bien e incubar durante 15 min a entre 2 y 8 ° C en el refrigerador. </li><li> Añadir 1-2 ml de PBS por 10 7 células para lavar y centrifugara 300 × g durante 10 min. </li><li> Aspirar el sobrenadante y resuspender completamente hasta 10 8 células en 500 l de PBS. </li></ol> 3. Selección magnética de subpoblaciones de leucocitos Nota: Una variedad de separadores de cuentas magnéticas y las columnas están disponibles para el aislamiento de la población específica de leucocitos por marcador de superficie celular de un número de fabricantes. El aislamiento puede ser por selección positiva basada en uno o unos pocos marcadores positivos o por selección negativa después de la depleción de las poblaciones no deseados. Un enfoque general de la realización de la selección positiva utilizando un marcador (es decir, CD4 de los linfocitos T helper, o CD14 de monocitos) se describe a continuación. <ol><li> Preparar y separador magnético prime incluyendo la columna de separación de acuerdo con las instrucciones del fabricante. </li><li> Aplicar tubo que contiene la muestra de PBMCs etiquetados. Proporcionar dos 15 ml tubos de centrifugación para la recogida del y unlabel marcado positivamentefracciones celulares ed, siguiendo las instrucciones del fabricante. </li><li> Contar el número de células 15 y resuspender los leucocitos seleccionados positivamente (es decir, linfocitos T CD4 + CD14 + o células) en medio completo de leucocitos, utilizando 1 ml de medio por 10 7 células. </li></ol> 4. CFSE tinción de leucocitos para la Evaluación de la proliferación Nota: CFSE ha sido ampliamente utilizado en las investigaciones inmunológicas, por tanto in vivo e in vitro. Nuestro protocolo ha sido optimizado para el estudio in vitro de MSC / PBMC (u otros leucocitos) interacciones. Si bien los pasos generales involucrados en la realización de marcaje in vitro CFSE son similares, puede haber diferencias en la dosis específica y el momento del protocolo 14. Esto puede ser debido a un número de factores, es decir, el tipo de célula específico utilizado, el número de células utilizadas-que probablemente puede afectar a la intensidad de la señal fluorescente CFSE. <ol&gt; <li> Diluir la solución CFSE madre (10 mM) en PBS a la concentración de trabajo deseada de 10 M (solución CFSE-trabajo). Preparar 10 7 células (es decir, PBMCs o células T) para el etiquetado con la solución CFSE-trabajo. </li><li> Centrifugar a 300 × g durante 10 min para obtener un sedimento de células y aspirar el sobrenadante. </li><li> Resuspender las células suavemente en 1 ml de solución (37 ° C) CFSE-trabajo precalentado y se incuban las células durante 10 minutos a 37 ° C. </li><li> Para lavar el exceso de CFSE, se diluye la suspensión celular con 10 veces (en volumen) de preenfriado (4 ° C) medio RPMI que contenía 10% de FBS. Sedimentar las células por centrifugación a 300 xg durante 5 min y descartar el sobrenadante. Lavar el sedimento celular de esta manera dos veces más. </li><li> Re-sedimentar las células por centrifugación y contar el número de células 15. Resuspender 10 7 células (ya sea PBMCs o células T) en 1 ml de medio completo de leucocitos precalentado fresco. </li></ol> 5. Co-cultura de MSC con leucocitos y activación de los leucocitos <ol><li> Medio Pre-caliente MSC completa (10% de FBS (previamente probado para un crecimiento óptimo MSC), 1% de L-glutamina, y 1% de penicilina / estreptomicina en medio DMEM de baja glucosa) a 37 ° C durante no más de 30 min. </li><li> MSCs semilla a 50.000 células en 1 ml de medio completo MSC en placas de 24 pocillos (densidad MSC: 25.000 células / cm 2) para la fijación O / N en una incubadora a 37 °, lo que permite que las células madre para alcanzar 80% de confluencia. </li><li> Medio Aspirar, y en base a los números de MSC sin semillas, añadir PBMC marcadas con CFSE (previamente marcado con consulte protocolo Paso 4 arriba) para las MSC cultivadas (sembradas el día anterior en placas de 24 pocillos) en 1 ml de medio completo de leucocitos en una 1:10 (proporción de células) Relación de co-cultivo de MSCs a PBMCs. </li><li> Añadir la fitohemaglutinina mitógeno (PHA), un activador de leucocitos no específica, a una concentración final de 10 mg / ml en un volumen total de 1 ml de medio completo por pocillo de leucocitos. <ol><li> AlternativEly, para estimular para la activación de los linfocitos T específicamente: utilizar alfa-CD3 / 28 microperlas y añadir a la de dos células de co-cultivo para obtener una relación de linfocitos bead-a-T de 1: 1. </li></ol></li><li> Para el control negativo, la placa de 500.000 PBMCs / pocillo (o una población específica de leucocitos efector; densidad celular: 250.000 leucocitos / cm 2) en una placa de 24 pocillos con 1 ml de medio completo de leucocitos solamente; para el control positivo, además de chapado en el mismo número de PBMC / pocillo, añadir PHA a una concentración final de 10 mg / ml. </li><li> En el tercero y quinto día del experimento de co-cultivo, evaluar la proliferación de leucocitos marcados con CFSE (colocados en tubos de fondo redondo) por análisis de citometría de flujo 17 con filtros adecuados para fluoresceína 488 nm de excitación y de emisión. Nota: tinción intracelular de citoquinas para el análisis de citometría de flujo también se puede realizar para estos leucocitos marcados con CFSE en este punto para evaluar los cambios en el perfil de expresión de citocinas de leucocitos como Modulated por MSC. Desde CFSE se evalúa con un apropiado filtro para fluoresceína, los anticuerpos seleccionados para evaluar diversas citoquinas necesitan ser conjugados a fluorocromos distintos de fluoresceína o espectro similar (es decir, ficoeritrina, peridinina clorofila complejo de proteína (PerCP)). </li></ol> 6. Variación: Ensayo Supresión Efector Magnetic Bead-seleccionado, MSC inducida inmunomoduladora leucocitos en Activado marcadas con CFSE células T efectoras CD4 + <ol><li> MSC semilla a 250.000 células en 3 ml de medio completo MSC en placas de 6 pocillos (densidad MSC: 25.000 células / cm 2) para la fijación O / N en una incubadora a 37 °, lo que permite a las células madre para llegar a 80% de confluencia. Al menos 3 placas de 6 pocillos son necesarios para garantizar PBMCs-MSC cocultured suficientes para la selección sub-población. </li><li> Aspirar medio, y agregue PBMCs separados según el Paso 1, pero en placas de 6 pocillos a 2,5 x 10 6 células / pocillo (densidad celular: 250 000 leukocytes / cm 2) con 3 ml de medio completo de leucocitos. Co-cultivo durante 48 a 72 horas en una incubadora a 37 °. </li><li> Magnética talón seleccionar población específica de leucocitos inmunomoduladores inducidos por el MSC (es decir, células CD14 +) como por las Secciones 2-3. Notas: tinción intracelular de citoquinas para el análisis de citometría de flujo se pueden realizar en este punto para evaluar los cambios en el perfil de expresión de citocinas de leucocitos, es decir, la expresión de la interleucina-10-como moduladas por MSCs. </li><li> Agregar CFSE marcado con células T CD4 + alogénicas generadas según las Secciones 2-4 en placas de 24 pocillos en 1 ml de leucocitos completa (densidad de células T: 250.000 células / cm 2) medio de leucocitos inducida por MSC talón-seleccionados en diversas proporciones , es decir, 1:10, 1: 5, 1: 2, y 1: 1 (una célula a otra) ratios. </li><li> Para estimular las células T CD4 +, añadir alfa-CD3 / 28 microperlas conjugados para obtener una proporción de grano a célula de 1: 1. </li><li> Para un control negativo, placa 500.000 células T CD4 + / pocillo en un (densidad de células T: 250.000 células / cm 2) placa de 24 pocillos con 1 ml de medio completo de leucocitos solamente; para el control positivo, además de chapado en el mismo número de células T CD4 + / pocillo, añadir alfa-CD3 / 28 microperlas conjugados para obtener una proporción de grano a célula de 1: 1. </li><li> En el día 3 rd de co-cultivo, evaluar la proliferación de las células T CD4 + marcadas con CFSE (colocados en tubos de fondo redondo) por el flujo de análisis de citometría de 16 con filtros apropiados para fluoresceína 488 nm de excitación y de emisión. </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 madre mesenquimatosas humanas (MSCs) son progenitores somáticas que pueden diferenciarse en los linajes mesodérmicos paraxiales de hueso, cartílago, y tejido adiposo 1-4, así como a unos pocos linajes extramesodermal 5. En primer lugar aislado de la médula ósea de adultos, estos progenitores multilinaje ahora se han encontrado en numerosos tejidos y 6-8, de forma inesperada, que se muestra a tener fuertes propiedades inmunomoduladoras que aparecen altamente susceptibles a la aplicación clínica 9-12. Mecanismos detallados implicados en los efectos inmunomoduladores activamente están siendo investigados por la aplicación efectiva de entidades patológicas específicas. Una de las formas más sencillas para evaluar la inmunomodulación es mediante la evaluación para la supresión de la proliferación de leucocitos efector 13. La mayoría de los leucocitos efectoras tales como linfocitos y monocitos T proliferan prodigiosamente cuando es estimulado o activado. La función inmunomoduladora se puede evaluar cuando la supresión de laproliferación se evidencia. Tradicionalmente, la proliferación de leucocitos efector ha sido evaluada por detección de [3 H] timidina en el ADN. Sin embargo, este método tiene inconvenientes significativos debido a las preocupaciones de la radiación y la post-uso disposición, así como el equipo complejo sea necesario. Si bien hay ensayos no radiactivos para evaluar la proliferación celular, el éster de succinimidilo de carboxifluoresceína (CFSE) de ensayo tiene otras ventajas tales como permitir que para la identificación de poblaciones celulares específicas, lo cual es especialmente útil en los experimentos de co-cultivo que implican múltiples tipos de células. CFSE es un colorante fluorescente celular que puede ser evaluado mediante análisis de citometría de flujo. Como las células se dividen, la intensidad de esta etiqueta celular se reduce proporcionalmente; Esto no sólo permite la determinación de la proliferación celular en general, pero también permite la evaluación del número de divisiones celulares hasta 8 divisiones antes de la fluorescencia se vuelve difícil en Detect contra la señal de fondo. Por otra parte, la estabilidad de la CFSE fluorescente permite el seguimiento in vivo de células marcadas tales que las células pueden ser visualizadas hasta muchos meses 14. Este ensayo también se puede variar para evaluar tipos específicos de leucocitos efectoras o la función inmunomoduladora de las poblaciones específicas de MSC inducida por leucocitos-tales inmunomoduladores como la interleucina-10 (IL-10) que produce monocitos CD14 + 15 mediante la realización de talón marcador de selección superficie magnética del poblaciones de células de interés antes o después de co-cultivo según sea apropiado. Nuestro protocolo describe el ensayo básico de la evaluación de los efectos inmunomoduladores de MSCs en los leucocitos efectoras (diagrama de flujo mostrado en la Figura 1) y una variación de este ensayo básico para la evaluación de la inducida por MSC inmunomodulación de leucocitos en alogénicas CD4 + efectoras linfocitos T (diagrama de flujo mostrado en la Figura 4).

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		<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.

La Figura 1 indica el esquema general del experimento, y la Figura 2 muestra el aspecto de las distintas condiciones de cultivo celular como se visualizaron mediante contraste de fase invertida microscopía. MSCs son células adherentes con una fibroblástica, la morfología en forma de huso, mientras que PBMCs y leucocitos son pequeñas células no adherentes redondas. Estos dos tipos de células morfológicamente diferentes pueden verse claramente en el co-cultivo. Al final del ensayo, cu...

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

Las propiedades inmunomoduladoras de las células madre mesenquimales humanas (MSC) aparecen cada vez más relevante para la aplicación clínica. El uso de un sistema de co-cultivo de MSCs y leucocitos de sangre periférica pre-manchado con la succinimidil éster colorante fluorescente carboxifluoresceína (CFSE), se describe la evaluación in vitro de MSC sobre la proliferación de leucocitos inmunomodulación efector y subpoblaciones específicas.

Evaluación de las propiedades inmunomoduladoras de las células madre mesenquimales humanas (MSC)

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 vistas.  National Health Research Institutes (NHRI).  Las propiedades inmunomoduladoras de las células madre mesenquimales humanas (MSC) aparecen cada vez más relevante para la aplicación clínica. El uso de un sistema de co-cultivo de MSCs y leucocitos de sangre periférica pre-manchado con la succinimidil éster colorante fluorescente carboxifluoresceína (CFSE), se describe la evaluación in vitro de MSC sobre la proliferación de leucocitos inmunomodulación efector y subpoblaciones específicas. 

Video: 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.

Expansión, purificación y evaluación funcional de la sangre periférica humana células NK

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

Investigación

Inmunología e Infección

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.

Evaluación cuantitativa de las células inmunes en el tejido de la médula espinal lesionada por citometría de flujo: un nuevo uso de un método de purificación 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 un nuevo enfoque para prevenir la infección: Preparación y<em&gt; In vitro</em&gt; Propiedades antimicrobianas de 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.

Desarrollo de derivados de células madre Las células T reguladoras específicas de antígeno contra la autoinmunidad

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.

Dextrano mejora la eficiencia de transducción lentivirales de las células NK primaria murinas y humanas

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 del agitador magnético para la detección de<em&gt; Trichinella</em&gt; Las larvas en muestras 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.

Un análisis de citotoxicidad basados en la citometría de flujo para la evaluación de la actividad de las células NK humanas

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.

Evaluación de la citotóxica y efectos inmunomoduladores de sustancias en lonchas de pulmón humano de precisión de corte

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.

Evaluación de la interfaz sináptica primaria humana de células de T de sangre periférica y tejido 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.

Diferenciación inducida de células similares a células M en monocapas de enteroide ileal derivadas de células madre humanas

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 ...