Este trabajo fue apoyado por el Centro de la Florida para la Investigación del tumor cerebral, el Preston A. Wells Jr. Centro de Terapia del tumor cerebral y los Institutos Nacionales de Salud (1R21CA141020-01).

1. Preparación de la suspensión Glioblastoma Single Cell <ol><li> Cultura Gliomasphere se establece y mantiene usando el ensayo de neurosphere (NSA) como se ha descrito previamente 2,11,12. </li><li> En el momento apropiado para pasar las gliomaspheres, el medio que contiene las esferas se retira, se colocaron en un tubo estéril de tamaño apropiado de cultivo de tejidos, y se centrifugaron a 800 rpm (110 g) durante 5 min a temperatura ambiente. </li><li> El sobrenadante se desechó y el sedimento de esferas se resuspendieron en 1 ml de 0,05% de tripsina-EDTA y se incubó a 37 ° C en un baño de agua durante 3-5 min. </li><li> Un volumen igual de inhibidor de tripsina de soja se utiliza a continuación para detener la actividad de la tripsina. </li><li> La suspensión de células se pipetearon suavemente hacia arriba y hacia abajo para asegurar la homogeneidad y la neutralización completa de la actividad de la tripsina. </li><li> La suspensión celular se centrifugó de nuevo a 800 rpm durante 5 min. Se elimina el sobrenadante y se resuspendieron las células en 1 ml de NeuroCultNSC Basal Medium. </li><li> 10μl de la suspensión de una sola célula se mezcla con 90μl de azul de tripano para realizar un recuento de células. </li></ol> 2. La tinción con célula de seguimiento Carboxyfluorescein Succinimidyl Ester (CFSE) tinte verde fluorescente <ol><li> Por cada 1 millón de células a teñir, 1 ml de reactivo de tinción se prepara mediante la adición de 1 l de 5 mM célula tinte CFSE Trace (Molecular Probes, Invitrogen) a 1 ml de NeuroCult Medio Basal NSC para dar una concentración final de tinción de 5 micras . </li><li> El reactivo de tinción que contiene las células se mezcla bien hasta homogeneidad y se incubaron a temperatura ambiente durante 10 min. </li><li> Durante la incubación, enfriado con hielo NeuroCult Medio Basal NSC se prepara como la solución de enfriamiento. </li><li> Para detener el proceso de tinción, aproximadamente 5-10 volúmenes de solución enfriamiento enfriado con hielo se añade a las células cargadas con CFSE. </li><li> La solución se centrifuga a 800 rpm durante 5 min y el sobrenadante se desecha. </li><li> En thes punto, debería aparecer un sedimento de células en la parte inferior del tubo con un tinte de color verde brillante de color, que indica la tinción fue exitosa. </li><li> Las células se lavaron por resuspensión del sedimento en 1-2 ml de medio fresco NeuroCult NSC Basal, centrifugando a 800 rpm durante 5 min y desechar el sobrenadante. Este procedimiento se repite para un total de tres lavados. </li><li> Después del tercer lavado, las células se resuspendieron en 1 ml de NeuroCult Medio Basal NSC y un recuento de células se realiza. </li><li> Las células teñidas con CFSE se siembran en matraces estériles de cultivo de tejidos a una densidad apropiada [50.000 células / ml] y se colocó en un 37 ° C / 5% CO2 humidificado incubadora. </li></ol> 3. CFSE Verificación de Carga <ol><li> Células chapada en cultivo NSA se puede supervisar bajo un microscopio fluorescente para verificar la tinción CFSE. En esta etapa, todas las células presentan color verde brillante (Figura 1). Alternativamente, una gota de la suspensión de células a partir deCélulas CFSE manchadas podría ser colocado en un portaobjetos de microscopio y se cubrió con un cubreobjetos para visualizar las células bajo el microscopio de fluorescencia. </li><li> CFSE etiquetado exitosa se ve confirmado por citometría de flujo. Tanto como 1-5 x 10 5 células de cada grupo es suficiente para la verificación CFSE tinción. El CFSE células cargadas y descargadas las células de control negativo se resuspendieron respectivamente en 1 x PBS-o NeuroCult Medio Basal NSC para un volumen total de 500 l y se colocaron en tubos FACS separados. </li><li> El citómetro de flujo se ajusta basándose en las instrucciones del manual de usuario para los parámetros relacionados. CFSE tiene un máximo de excitación de longitud de onda de 492 nm y un máximo de emisión de 517 nm. </li><li> En primer lugar, las células de control sin carga se ejecutan y los eventos adquiridos se registran en la dispersión frontal y lateral (FSC frente a SSC) y histogramas, y la población de células principal está cerrado (Figura 2A). </li><li> Del mismo modo, las células cargadas con CFSE se analizaron usando el mismo parámetros utilizado para las células control (Figura 2B). </li></ol> 4. El aislamiento de la lenta dividiendo [CFSE es decir Retención] Población celulares utilizando Clasificación celular activada por fluorescencia (FACS) <ol><li> Tras la división, la intensidad de CFSE se disminuyeron y las células hGBM formar gliomaspheres que se componen de células que muestran diferentes niveles de intensidad de fluorescencia verde (Figura 3). Cuando los gliomaspheres han alcanzado un tamaño medio de aproximadamente 150-200 micras de diámetro (aproximadamente de 5 a 10 días de carga CFSE mensaje en función de la tasa de crecimiento de células de línea), el medio que contiene las esferas se retira, se colocaron en un cultivo de tejido estéril tamaño apropiado tubo, y se centrifugó a 800 rpm durante 5 min a temperatura ambiente. </li><li> Una única suspensión de células se prepararon como se describe en los pasos anteriores y las células se resuspendieron en PBS para un recuento de células con el fin de preparar una suspensión celular con una densidad de hasta 10 7 células por mililitro. </li><li> En popaer resuspender las células en un volumen apropiado de PBS, yoduro de propidio (PI, Sigma, 500 mg / ml) se añade a una concentración de 1 l / ml de suspensión celular a excluir las células muertas cuando se ordenan / analizadas por citometría de flujo. </li><li> Dos tubos estériles de 15 ml de cultivo de tejidos que contienen cada uno 1 ml de medio completo NeuroCult NSC se utiliza para recoger las células se dividen según lento (células CFSE retención) y las células que se dividen rápidamente las células CFSE (dilución). </li><li> En primer lugar, la suspensión de células a partir del grupo de células carga y se ejecuta a través del citómetro de flujo. </li><li> Las células (eventos) se trazan sobre la base de dispersión frontal (FSC) frente a dispersión lateral (SSC) y los parámetros de voltaje para ambos parámetros se ajustan de modo que la mayoría de las células están incluidas en la trama de flujo. </li><li> Para excluir las células muertas o dañadas, las células se trazan sobre la base de la reactividad frente a SSC PI y la única población de células vivas (PI negativo) es cerrada. </li><li> Entonces, una población celular homogénea se selecciona basándose en forward dispersión (FSC) frente a dispersión lateral (SSC). </li><li> La única población homogénea de células vivas se traza en un histograma basado en la frecuencia de células frente a la intensidad de CFSE establecido en una escala logarítmica. La intensidad del láser CFSE se ajusta con el fin de colocar la señal negativa entre 0 y 100. </li><li> Usando los mismos parámetros y una estrategia de activación periódica similar, las células CFSE etiquetados se ejecutan a través del citómetro de flujo y se analizó (Figura 4C). </li><li> Posteriormente, una población lento división de las células y la población en general (más rápido las células en división) se identifican basándose en su capacidad para retener CFSE (CFSE alto superior al 5% y CFSE bajo de fondo 85%, respectivamente). </li><li> Estas poblaciones de células diferentes se clasifican en distintas 15 ml tubos estériles de cultivo de tejidos que contienen 1 ml de medio completo NSC. </li><li> Las células clasificadas se centrifugan a 800 rpm durante 5 min. </li><li> El sobrenadante se desechó y el sedimento se resuspendió está en una aprovolumen de medio de apropiados (en función del número de eventos aislados o tamaño de los gránulos) y un recuento de células se lleva a cabo. </li><li> Las células de las diferentes poblaciones según luego se sembraron en placas a 50.000 células por mililitro en un matraz estéril apropiado de cultivo de tejido y se colocan en un 37 ° C / 5% CO2 humidificado incubadora durante 5-10 días antes de que se pasa en serie o utilizados para experimentos posteriores. </li></ol> 5. Los resultados representativos Después de la carga CFSE, todas las células presentan una fluorescencia de color verde intenso como se visualizaron bajo el microscopio (Figura 1). Este color verde-teñido se puede ver incluso con el ojo desnudo (ver el video). Analizando estas células con citometría de flujo también muestra la fluorescencia de color verde muy intenso en comparación con las células control (Figura 2). Una vez cargadas las células CFSE enchapado en cultivo neurosphere, estas células proliferan y forman 150-200 micras gliomaspheres en 5-10 días. Como las células proliferan, t<html> colorante fluorescente que se distribuye por igual entre las células hijas a divisiones, llevando a la reducción a la mitad de la intensidad de la fluorescencia con cada división celular. Los glioblastomas son funcionalmente heterogéneo y se componen de células que proliferan en escalas de tiempo diferentes. El método descrito en este protocolo permite identificar y aislar las células en división rápida y lenta en virtud de su capacidad de diluir o retener CFSE, respectivamente (Figura 3, también ver el video). Análisis de citometría de flujo de gliomaspheres disociadas generados a partir de células cargadas con CFSE en comparación con las células sin carga se muestra diversas propiedades de retención de CFSE (Figura 4). Fast células en división (inferior al 85%) o lentas las células en división (el 5% - CFSE bajo) gliomapshere exhibición capacidad de formación cuando se cultivan en las condiciones del ensayo neurosphere (Figura 5). gura 1 &quot;/&gt; Figura 1. Las células disociadas tumorales inmediatamente después de la carga con el colorante CFSE. Las células presentan color verde brillante demostración de etiquetado éxito. Aumento original; 10 x. <img src="/files/ftp_upload/3918/3918fig2.jpg" alt="Figura 2" /> Figura 2. Confirmación CFSE carga usando citometría de flujo. A) células sin carga, B) CFSE cargado células y c) comparar la intensidad de fluorescencia de CFSE en las células cargadas frente descargada. Tenga en cuenta que hay varios órdenes de magnitud de diferencia en la intensidad de fluorescencia entre las células marcadas y no marcadas. <img src="/files/ftp_upload/3918/3918fig3.jpg" alt="Figura 3" /> Figura 3. Un representante gliomasphere derivada de una célula CFSE-cargado 6 días después de la siembra. Algunas células exhiben tinción CFSE muy brillante. Aumento original, 63 x. <img src = &quot;/ files/ftp_upload/3918/3918fig4.jpg&quot; alt = &quot;Figura 4&quot; /&gt; Figura 4. Representante de citometría de flujo parcelas / histogramas para el aislamiento de las células en división rápida y lenta de 6 días después de la tinción con CFSE. Una reactividad) FSC frente a SSC para mostrar toda la población celular, B) vs SSC PI para excluir las células muertas o dañadas, C) FSC frente a SSC para puerta de una población homogénea de células vivas, D) demuestra la presencia de células CFSE retención 6 días después de CFSE etiquetado como se evidencia por una mayor intensidad de fluorescencia en comparación con el control negativo. El histograma también muestra que la intensidad de CFSE decayó con el tiempo. E) Histograma que muestra la estrategia de activación periódica para identificar células CFSE retención (superior al 5%) y las células CFSE de dilución (85% inferior). <img src="/files/ftp_upload/3918/3918fig5.jpg" alt="Figura 5" /> Figura 5. Gliomaspheres representativos generados a partir de <stronG&gt; A) rápida división de las células (CFSE bajo) y células B) slow-división (CFSE alto). Aumento original:. 10x y 20x respectivamente C) Número de Representante celular obtenida a partir de rápido o lento división celular derivada de cultivo en el tiempo paso. P &lt;0,05, t-test. <a href="http://www.jove.com/files/ftp_upload/3918/3918fig5large.jpg" target="_blank">Haga clic aquí para ampliar la cifra</a> .

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No hay conflictos de interés declarado.

Un creciente número de estudios han demostrado la importancia de una lenta dividiendo sub-compartimiento de células en el cáncer que contribuyen a la formación de tumores y la resistencia al tratamiento 2-8. Por lo tanto es crítico para describir y estandarizar los métodos experimentales que permiten el estudio de esta subpoblación específica de células o de manera más general para comparar las subpoblaciones con diferentes tasas de crecimiento. Usando CFSE para identificar y aislar sub-fracciones d...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Tipo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> NeuroCult NSC Basal Medium (Humana) </td><td> Medio </td><td> Stem Cell Technologies </td><td> 05750 </td></tr><tr><td> NeuroCult NSC proliferación Suplementos (Humana) </td><td> Suplemento Media </td><td> Stem Cell Technologies </td><td> 05753 </td></tr><tr><td> 0,05% de tripsina-EDTA </td><td> Reactivo </td><td> Gibco </td><td> 25300-062 </td></tr><tr><td> Inhibidor de tripsina de soja </td><td> Reactivo </td><td> Sigma </td><td> T6522 </td></tr><tr><td> Penicilina / Estreptomicina </td><td> Reactivo </td><td> Gibco </td><td> 15140-122 </td></tr><tr><td> Matraz T25 </td><td> Cultura cerámica </td><td> Nalge Nunc internacional </td><td> 136196 </td></tr><tr><td> T80 frasco </td><td> Cultura cerámica </td><td> Nalge Nunc internacional </td><td> 178905 </td></tr><tr><td> 15 ml tubos </td><td> Cultura cerámica </td><td> BD Falcon </td><td> 352096 </td></tr><tr><td> 50 ml tubos </td><td> Cultura cerámica </td><td> BD Falcon </td><td> 352070 </td></tr><tr><td> FEAG </td><td> Factor de crecimiento </td><td> De I + D </td><td> 2028-EG </td></tr><tr><td> CFSE CellTrace Proliferación Celular Kit </td><td> División Celular fluorescente seguimiento Dye </td><td> Molecular Probes, Invitrogen </td><td> C34554 </td></tr></tbody></table>

identification and isolation of slow-dividing cells in human glioblastoma using carboxy fluorescein succinimidyl ester (cfse)

Heterogeneidad tumoral representa una característica fundamental de apoyo robustez tumor y presenta un obstáculo fundamental para el desarrollo de estrategias terapéuticas 1. Para superar el problema de la heterogeneidad del tumor, es esencial para desarrollar ensayos y herramientas que permiten fenotípica, (epi) la identificación genética y funcional y la caracterización de las subpoblaciones tumorales que conducen a enfermedades y patologías específicas representan objetivos clínicamente relevantes. Actualmente está bien establecido que los tumores presentan distintas sub-fracciones de células con diferentes frecuencias de la división celular, y que los criterios funcionales de ser lento bicicleta está positivamente asociado con la capacidad de formación de tumores en varios tipos de cáncer, incluidos los del cerebro, mama, piel y páncreas, así como leucemia 2-8. El colorante fluorescente carboxifluoresceína succinimidil éster (CFSE) se ha utilizado para el seguimiento de la frecuencia de división de las células in vitro e in vivo en transmisión sanguínea tumors y tumores sólidos tales como el glioblastoma 2,7,8. La célula-permeante no fluorescente pro-fármaco de la CFSE se convierte mediante esterasas intracelulares a un compuesto fluorescente, la cual es retenida dentro de las células por unión covalente a las proteínas mediante la reacción de su radical succinimidilo con grupos amina intracelulares para formar enlaces amídicos estables 9. El tinte fluorescente se distribuye por igual entre las células hijas a divisiones, llevando a la reducción a la mitad de la intensidad de la fluorescencia con cada división celular. Esto permite realizar un seguimiento de la frecuencia del ciclo celular hasta ocho a diez rondas de división 10. Capacidad de retención de CFSE se utilizó con las células tumorales del cerebro para identificar y aislar una subpoblación bicicleta lenta (parte superior 5% de retención de colorante células) demostraron ser enriquecido en actividad de células madre de cáncer 2. Este protocolo describe la técnica de tinción de las células con CFSE y el aislamiento de las poblaciones individuales dentro de una cultura de humano glioblastoma (GBM)-derivados de células que poseen diferentes tipos de división usando citometría de flujo 2. La técnica ha servido para identificar y aislar un tumor cerebral lento bicicleta población de células en virtud de su capacidad para retener la CFSE etiquetado.

Watch this Scientific Journal Video about Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE at JoVE.com

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Este protocolo de video demuestra la aplicación de el colorante fluorescente carboxifluoresceína succinimidil éster (CFSE) para la identificación y separación de los diferentes sub-poblaciones de células de glioblastoma humano en base de la frecuencia de la división celular.

La identificación y aislamiento de Slow división de las células de glioblastoma humano utilizando carboxi fluoresceína Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

identification-isolation-slow-dividing-cells-human-glioblastoma-using

Research

JoVE Journal

Medicine

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

14.0K vistas.  The University of Florida. Este protocolo de video demuestra la aplicación de el colorante fluorescente carboxifluoresceína succinimidil éster (CFSE) para la identificación y separación de los diferentes sub-poblaciones de células de glioblastoma humano en base de la frecuencia de la división celular.

Video: Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Investigación

Medicina

Isolation of Cancer Stem Cells From Human Prostate Cancer Samples

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly isolated from human tissues and serially propagated in immunodeficient mice, typically through antibody labeling of subpopulations of cells and fractionation by flow cytometry. However, the unique clinical features of prostate cancer have considerably limited the study of prostate CSCs from fresh human tumor samples. We recently reported the isolation of prostate CSCs directly from human tissues by virtue of their HLA class I (HLAI)-negative phenotype. Prostate cancer cells are harvested from surgical specimens and mechanically dissociated. A cell suspension is generated and labeled with fluorescently conjugated HLAI and stromal antibodies. Subpopulations of HLAI-negative cells are finally isolated using a flow cytometer. The principal limitation of this protocol is the frequently microscopic and multifocal nature of primary cancer in prostatectomy specimens. Nonetheless, isolated live prostate CSCs are suitable for molecular characterization and functional validation by transplantation in immunodeficient mice.

The isolation of cancer stem cells (CSCs) directly from human tissues is requisite for their biological characterization. This manuscript describes a methodology for the isolation of prostate CSCs from human tissues, while also providing tips on troubleshooting difficult steps.

El aislamiento de las células madre del cáncer de muestras de cáncer de próstata humano

The cancer stem cell (CSC) model has been considerably revisited over the last two decades. During this time CSCs have been identified and directly ...

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

El aislamiento, la criopreservación y cultivo de células epiteliales Amnion Humanos para Aplicaciones clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

Isolation of Human Lymphatic Endothelial Cells by Multi-parameter Fluorescence-activated Cell Sorting

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity but little is known about their biology. Isolating highly pure human lymphatic endothelial cells (LECs) from diseased and healthy tissue would facilitate studies of the lymphatic endothelium at genetic, molecular and cellular levels. It is anticipated that these investigations may reveal targets for new therapies that may change the clinical management of these conditions. A protocol describing the isolation of human foreskin LECs and lymphatic malformation lymphatic endothelial cells (LM LECs) is presented. To obtain a single cell suspension tissue was minced and enzymatically treated using dispase II and collagenase II. The resulting single cell suspension was then labelled with antibodies to cluster of differentiation (CD) markers CD34, CD31, Vascular Endothelial Growth Factor-3 (VEGFR-3) and PODOPLANIN. Stained viable cells were sorted on a fluorescently activated cell sorter (FACS) to separate the CD34LowCD31PosVEGFR-3PosPODOPLANINPos LM LEC population from other endothelial and non-endothelial cells. The sorted LM LECs were cultured and expanded on fibronectin-coated flasks for further experimental use.

The goal of this protocol is to isolate lymphatic endothelial cells lining human lymphatic malformation cyst-like vessels and foreskins using fluorescence-activated cell sorting (FACS). Subsequent cell culturing and expansion of these cells permits a new level of experimental sophistication for genetic, proteomic, functional and cell differentiation studies.

Aislamiento de células endoteliales linfático humano por Multi-parámetro de clasificación de células activadas por fluorescencia

Lymphatic system disorders such as primary lymphedema, lymphatic malformations and lymphatic tumors are rare conditions that cause significant morbidity ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

El estudio de cáncer de páncreas Tallo Características de las células para el desarrollo de nuevas estrategias de tratamiento

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

Generación de microtumores Uso Humano 3D Biogel sistema de cultivo y las células de glioblastoma derivados de los pacientes para Kinomic de perfiles y pruebas de respuesta a los fármacos

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

El agotamiento de las células de ratón a partir de xenoinjertos de tumores humanos mejora significativamente análisis de aguas abajo de las células diana

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Intranasal Delivery of Therapeutic Stem Cells to Glioblastoma in a Mouse Model

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of therapeutic stem cells via the intranasal route is a recently discovered alternative strategy, with strong potential for clinical translation, due to its non-invasive nature compared to intracranial implantation or delivery via systemic routes. The lack of blood brain barrier further strengthens the therapeutic potential of stem cells undergoing intranasal brain entry. This article summarizes the essential techniques utilized in our studies and outlines the basic principles of intranasal strategy for stem cell delivery using a mouse model of intracranial glioma xenografts. We demonstrate the optimized procedures that generate consistent and reproducible results with specific predetermined experimental parameters and offer guidelines for streamlined work flow that ensure efficient execution and reliable experimental outcome. The article is designed to serve as a baseline for further experimental customization based on hypothesis, stem cell types, or tumor specifics.

Stem cells are promising therapeutic carriers to treat brain tumors due to their intrinsic tumor tropism. Non-invasive intranasal stem cell delivery bypasses the blood brain barrier and demonstrates strong potential for clinical translation. This article summarizes the basic principles of intranasal stem cell delivery in a mouse model of glioma.

Entrega intranasal de células madre terapéuticas a glioblastoma en un modelo de ratón

The intrinsic tropism towards brain malignancies renders stem cells as promising carriers of therapeutic agents against malignant tumors. The delivery of ...

Characterization of Immune Cells in Human Adipose Tissue by Using Flow Cytometry

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the development of obesity-associated complications such as type 2 diabetes. To quantitatively and qualitatively investigate the immune cell subsets in human AT deposits, we have developed a flow cytometry approach. The stromal vascular fraction (SVF), containing the immune cells, is isolated from subcutaneous and visceral AT biopsies by collagenase digestion. Adipocytes are removed after centrifugation. The SVF cells are stained for multiple membrane-bound markers selected to differentiate between immune cell subsets and analyzed using flow cytometry. As a result of this approach, pro- and anti-inflammatory macrophage subsets, dendritic cells (DCs), B-cells, CD4+ and CD8+ T-cells, and NK cells can be detected and quantified. This method gives detailed information about immune cells in AT and the amount of each specific subset. Since there are numerous fluorescent antibodies available, our flow cytometry approach can be adjusted to measure various other cellular and intracellular markers of interest.

This article describes a method to analyze immune cell content of adipose tissue by isolation of immune cells from adipose tissue and subsequent analysis using flow cytometry.

Caracterización de células inmunes en el tejido adiposo humano mediante el uso de citometría de flujo

Infiltration of immune cells in the subcutaneous and visceral adipose tissue (AT) deposits leads to a low-grade inflammation contributing to the ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Aislamiento de células Decidual humanas primarias de las membranas fetales de la placenta de término

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...