Serene Samyesudhas and Dr. Lynn Roy assisted in preparing samples for filming.

1. Cultivo Celular y Marcaje fluorescente de líneas celulares de cáncer de ovario <ol><li> Preparar medios SKOV3: medios McCoy suplementado con 10% de suero bovino fetal (FBS), 0,1 mM de L-glutamina, 50 U / ml de penicilina, y 50 mg / ml de estreptomicina. Mantener OVCA429 células en medio mínimo esencial (MEM) suplementado con 10% FBS, piruvato sódico 1 mM, 0,1 mM de L-glutamina, 50 U / ml de penicilina y 50 mg / ml de estreptomicina. </li><li> Propagar las líneas celulares SKOV3 y OVCA429 en un incubador humidificado a 37 ° C con 5% de CO 2. Se cultivan las células a 40-50% de confluencia. </li><li> Generar células que expresan la proteína fluorescente roja (RFP) con el fin de controlar la viabilidad in vitro e in vivo. <ol><li> Para generar células que expresan SKOV3 RFP, añadir partículas lentivirales que expresan RFP y 10 g / ml de polibreno a los medios SKOV3 en ausencia de penicilina y estreptomicina durante 72 horas. </li><li> Después de 72 horas, seleccione para RFP células positivas por fluorescencia de células activado Sorting (FACS) como sigue: trypsinize RFP y no las células que expresan RFP, centrifugar a 125 xg durante 5 min, se resuspenden las células en 10 7 células / ml en PBS que contenía 0,1% de BSA. En un clasificador de células, usar células no RFP para determinar los parámetros de clasificación. Luego recoger células que expresan altos RFP usando un láser de 561 nm. </li><li> Alternativamente, si el plásmido lentiviral expresa un casete de resistencia a los antibióticos, seleccionar las células que expresan RFP mediante el cultivo en el antibiótico apropiado (por ejemplo, 10 mg / ml de blasticidina). NOTA: La cantidad de partículas lentivirales que necesita ser usada puede variar por lote y por tipo de célula. Lentiviral título adecuado debe determinarse para cada tipo de célula. </li></ol></li></ol> 2. enriquecimiento de las células madre del cáncer <ol><li> Tratar SKOV3, SKOV3-RFP, o OVCA429 líneas celulares con 20 M de cisplatino durante 72 horas. PRECAUCIÓN: El cisplatino es tóxico. Use ropa de protección / guantes y evitar el contacto con la piel y las membranas mucosas. No se deshaga de cisplatino in de aguas residuales. </li><li> Preparar medios CSC: suero libre de medio DMEM / F12 suplementado con 5 mg / ml de insulina, 10 ng / factor de crecimiento epidérmico humano recombinante ml (EGF), 10 ng / factor de crecimiento de fibroblastos básico ml (bFGF), 12 ng / ml del factor inhibidor de la leucemia , y 0,4% de albúmina de suero bovino (BSA). </li><li> Células trypsinize. Cultura células supervivientes en los medios de CSC. NOTA: Después de 2 días en cultivo esferoides no adherentes se formarán. </li><li> Cambio de medio cada 2 días mediante la recopilación de los medios de comunicación que contiene las células y centrifugar a 125 xg durante 5 min. Entonces resuspender el precipitado en los medios de comunicación CSC fresco. Compruebe las células para la viabilidad cada 2 días contando las células y la realización de exclusión con azul de tripano. </li><li> Luego recoger CSC para experimentos adicionales por centrifugación 129 xg durante 5 min y resuspender el sedimento en fenol que contiene tiocianato de guanidinio (ARN), solución salina tamponada con fosfato (PBS: NaCl 137 mM, KCl 2,7 mM, y 11,9 mM de tampón de fosfato, pH 7,4) con 0,1% de BSA (por citometría de flujo), o los medios de comunicación CSC para continuarcultivar las células. </li><li> Controle la morfología celular CSC utilizando un microscopio óptico a 20x y 40x aumentos. </li></ol> 3. CSC Caracterización mediante análisis de expresión génica de marcadores de células madre <ol><li> Recoger 3 preparaciones de células madre cancerosas mediante la recopilación de medios que contienen células, centrifugar a 129 xg durante 5 min, y resuspender el precipitado en una solución de fenol que contiene tiocianato de guanidinio (otros métodos para la extracción de ARN se pueden utilizar). </li><li> Preparar el ARN total usando TRIzol según el protocolo del fabricante. Sintetizar ADN complementario (ADNc) 0,1-1 g de ARN usando un kit de transcripción inversa de ADNc disponibles comercialmente. </li><li> Realizar transcripción inversa reacción en cadena de la polimerasa cuantitativa (QRT-PCR). <ol><li> Maquillaje mezcla maestra para cada conjunto de cebadores que contenían la mezcla de QRT-PCR, cebadores, y agua a un total de 8 l por muestra (cebadores con diferentes fluoróforos se pueden amplificar en el mismo pocillo). Por ejemplo, generaruna mezcla maestra para Oct4-FAM, Nanog-Hex, y GAPDH-CY5. Generar mezclas maestras separadas para Nestin y PROM1. Añadir 2 l de cDNA plantilla por muestra. NOTA: Para este estudio, QRT-PCR se realizó usando los cebadores listados en la Tabla 1. </li><li> Realizar todos los QRT-PCR por duplicado para cada muestra utilizando un termociclador de PCR en tiempo real capaz de detectar múltiples fluoróforos. Incluya la plantilla no controles. </li><li> Normalizar la expresión génica de células madre para la expresión de GAPDH en cada muestra usando el método ΔΔC T. </li></ol></li></ol><table border="0" cellpadding="0" cellspacing="0" width="746"><colgroup><col /><col /></colgroup><tbody><tr height="21"><td height="63" rowspan="3" style="height:62px;width:111px;"> Oct-4-FAM </td><td style="width:636px;"> Cebador 1: 5&#39;-CCCAAGGAATAGTCTGTAGAAGTG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Primer 2: 5&#39;-TGCATGAGTCAGTGAACAGG-3 &#39;</td></tr><tr height="21"><td height="21" style="height:21px;"> FAM: 5&#39;-CTTCCAAGC / ZEN / TGCCCACCTAACTTCT / 3IABkFQ -3 &#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> Nanog-HEX </td><td> Cebador 1: 5&#39;-CCTTCTGCGTCACACCATT-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Primer 2: 5&#39;-AACTCTCCAACATCCTGAACC-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Sonda HEX: 5&#39;-CTGCCACCT / ZEN.CTTAGATTTCATTCTCTGGT / 3IABkFQ-3 &#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> GAPDH con Cy5 </td><td> Cebador 1: 5&#39;-TGTAGTTGAGGTCAATGAAGGG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Primer 2: 5&#39;-ACATCGCTCAGACACCATG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Sonda CY5: 5&#39;-AAGGTCGGAGTCAACGGATTTGGTC / 3IABRQSP-3 &#39; </td><html></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> Nestin-FAM </td><td> Cebador 1: 5&#39;-AGGACCTGAGCGATCTGG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Primer 2: 5&#39;-CGTTGGAACAGAGGTTGGAG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> FAM: 5&#39;-AACTTTTCA / ZEN / GTAGCCCGCAGCC / 3IABkFQ -3 &#39; </td></tr><tr height="21"><td height="63" rowspan="3" style="height:62px;"> CD133-HEX </td><td> Cebador 1: 5&#39;-ACTCTCTCCAACAATCCATTCC-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Primer 2: 5&#39;-AAACAATTCACCAGCAACGAG-3 &#39; </td></tr><tr height="21"><td height="21" style="height:21px;"> Sonda HEX: 5&#39;-ACAATCACT / ZEN / GAGCACTCTATACCAAAGCG / 3IABkFQ-3 </td></tr></tbody></table> Cuadro 1 Primers utilizados para la caracterización CSC por QRT-PCR. e_title &quot;&gt; 4. CSC Análisis de Superficie Celular marcadores CD117 y CD133 <ol><li> CSC cosecha mediante la recopilación de los medios de comunicación que contiene las células y centrifugar a 125 xg durante 5 min. Añadir 500 l de una solución desprendimiento de células no proteolítica para romper las células-up. Centrifugar las células a 125 xg durante 5 min para eliminar la solución desprendimiento de las células. A continuación, lavar las células dos veces con PBS frío. </li><li> Incubar en medio de cultivo normal durante 1 hora antes de células de etiquetado. Centrifugar las células durante 5 min a 129 x g. Resuspender las células en PBS con 0,1% de BSA con anti-CD133-PE y anti-CD117-FITC. </li><li> Fijar las células durante la noche en paraformaldehído al 1%. Precaución: El paraformaldehido es tóxico (carcinógeno). Hacer en campana de extracción y utilizar dentro de una semana. </li><li> Realizar citometría de flujo para analizar CD117 (con un anti-CD117 conjugado a FITC) y CD133 (anti-CD133 con conjugado a PE) expresión en la superficie celular en un citómetro de flujo. Recopilar datos sobre las células en el FL1 (FITC) y FL3 (PE) para los canales. Generar puertas para las células que expresan tantoFITC y PE. Generar un gráfico de puntos con 4 cuadrantes (FITC y PE, FITC + PE, PE FITC +, y + FITC y PE +) y determinar el número de células en cada cuadrante. </li></ol> 5. Analizando CSC para la respuesta terapéutica <ol><li> Placa de 5.000 células por pocillo (SKOV3, SKOV3-RFP, SKOV3 CSC y SKOV3-RFP CSC) en placas de 96 pocillos durante la noche. </li><li> Tratar con cisplatino (0, 1, 10, 100, y 1.000 M) o paclitaxel (0, 0,01, 0,1, 1, 10 y 100 mM) durante 96 h. </li><li> Realizar MTT (3 (4,5-dimetiltiazol-2-il) -2,5-difeniltetrazolio) ensayo de viabilidad celular. <ol><li> Placa de 5000 células por 100 l por pocillo en una placa de 96 pocillos (células de la placa por duplicado y los medios de comunicación incluyen sólo los pocillos de control). </li><li> Después de 24 horas, añadir 10 l de 10 mg / ml de MTT. Incubar a 37 ° C durante 2-4 h (hasta que se forma un precipitado). </li><li> Añadir 100 l de HCl mM MTT disolvente 4, 0,1% de NP-40 en isopropanol. Incubar durante 15 min en la oscuridad. </li><li> Leer las placas a 570 nm en un plato reader. </li></ol></li></ol>

<ol>
	<li>Pennington, K., Pulaski, H., Pennington, M., Liu, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Too+Much+of+a+Good+Thing:+Suicide+Prevention+Promotes+Chemoresistance+in+Ovarian+Carcinoma.">Too Much of a Good Thing: Suicide Prevention Promotes Chemoresistance in Ovarian Carcinoma.</a> Curr Cancer Drug Targets. 10, 575-583 (2010).</li><li>Thigpen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rational+approach+to+the+management+of+recurrent+or+persistent+ovarian+carcinoma.">A rational approach to the management of recurrent or persistent ovarian carcinoma.</a> Clin Obstet Gynecol. 55, 114-130 (2012).</li><li>Burgos-Ojeda, D., Rueda, B. R., Buckanovich, R. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+cancer+stem+cell+markers:+prognostic+and+therapeutic+implications.">Ovarian cancer stem cell markers: prognostic and therapeutic implications.</a> Cancer letters. 322, 1-7 (2012).</li><li>Ma, L., Lai, D., Liu, T., Cheng, W., Guo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem-like+cells+can+be+isolated+with+drug+selection+in+human+ovarian+cancer+cell+line+SKOV3.">Cancer stem-like cells can be isolated with drug selection in human ovarian cancer cell line SKOV3.</a> Acta Biochim Biophys Sin (Shanghai). 42, 593-602 (2010).</li><li>Bonnet, D., Dick, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+acute+myeloid+leukemia+is+organized+as+a+hierarchy+that+originates+from+a+primitive+hematopoietic+cell.">Human acute myeloid leukemia is organized as a hierarchy that originates from a primitive hematopoietic cell.</a> Nature Medicine. 3, 730-737 (1997).</li><li>Alison, M. R., Lim, S. M., Nicholson, L. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer+stem+cells:+problems+for+therapy.">Cancer stem cells: problems for therapy.</a> The Journal of pathology. 223, 147-161 (2011).</li><li>Luo, X., Dong, Z., Chen, Y., Yang, L., Lai, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+ovarian+cancer+stem-like+cells+is+associated+with+epithelial+to+mesenchymal+transition+through+an+miRNA-activated+AKT+pathway.">Enrichment of ovarian cancer stem-like cells is associated with epithelial to mesenchymal transition through an miRNA-activated AKT pathway.</a> Cell proliferation. 46, 436-446 (2013).</li><li>Wang, L., Mezencev, R., Bowen, N. J., Matyunina, L. V., McDonald, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+stem-like+cells+from+a+human+ovarian+cancer+cell+line.">Isolation and characterization of stem-like cells from a human ovarian cancer cell line.</a> Molecular and cellular biochemistry. 363, 257-268 (2012).</li><li>Abubaker, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+single+treatment+of+chemotherapy+results+in+the+enrichment+of+ovarian+cancer+stem+cell-like+cells+leading+to+an+increased+tumor+burden.">Short-term single treatment of chemotherapy results in the enrichment of ovarian cancer stem cell-like cells leading to an increased tumor burden.</a> Molecular cancer. 12, 24 (2013).</li><li>Conic, I., Dimov, I., Tasic-Dimov, D., Djordjevic, B., Stefanovic, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ovarian+epithelial+cancer+stem+cells.">Ovarian epithelial cancer stem cells.</a> ScientificWorldJournal. 11, 1243-1269 (2011).</li><li>Liu, K. 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The authors declare they have no competing financial interest.

CSC que son resistentes a la terapia pueden ser responsables de la recaída después del tratamiento del tumor primario. Caracterización de las CSC puede conducir a mejores tratamientos para el cáncer de ovario. Los parámetros críticos en el establecimiento de las CSC quimiorresistentes utilizando el protocolo anterior son los plazos de la duración del tratamiento con quimioterapia y la concentración de la quimioterapia. Cuando se utiliza el protocolo de Ma et al. se encontró que después de 7 días de t...

Resistance to chemotherapy is a major impediment to the treatment and cure of cancer. Ovarian cancer is the 5th leading cause of cancer-related deaths among women in the United States (American Cancer Society Facts and Figures 2013). Patients initially respond well to chemotherapy, but most patients relapse1. After relapse patients are treated with a variety of additional chemotherapy agents with very little benefit2. General properties of CSCs include unlimited proliferative capabilities, retention of an undifferentiated state, resistance to drug treatment, efficient DNA repair, and ability to generate malignant tumor cells with different phenotypes3. CSCs frequently exhibit expression of stem cell genes such as Nanog, Oct4, Sox2, Nestin, CD133, and CD117. CSCs often express elevated levels of ABCG2, and ALDH genes that may contribute to drug efflux and metabolism3,4. 
The first definitive evidence for CSCs was demonstrated by isolating acute myeloid leukemia-initiating cells that were capable of self-renewal and tumor generation5. These leukemic stem cells expressed surface CD34 and generated leukemia in NOD/SCID (immunocompromised) mice5. Since then CSCs have been identified in many cancer types including leukemias/lymphomas, breast, bladder, colorectal, endometrial, sarcomas, hepatocellular carcinoma, melanomas, gliomas, ovarian, pancreatic, prostate, squamous cell carcinoma, and lung6. Therefore, being able to study this subtype of cancer cell is advantageous.
The goal of this study is to create a protocol for the selection and isolation of chemoresistant CSCs. Several methods have been reported for the isolation of CSCs from ovarian cancer cell lines. Non-adherent spheroids isolated from OVCAR-3, SKOV3, or HO8910 cultures demonstrate stem-like properties7,8. Isolation of CD133+ cells from OVCAR-3 cultures also yields CSCs. CSCs have also been selected in culture by treatment with chemotherapeutic agents. Treating tumor cell lines (OVCA433, Hey, and SKOV3 cells) with cisplatin and paclitaxel allows for the expansion and isolation of CSCs4,9. While culture of some cell types in CSC media leads to isolation of CSCs, SKOV3 cells did not survive culture in serum-free media or form sphere cells4. Therefore, treatment of cells with cisplatin and paclitaxel aided the expansion or isolation of this population4.
Using a modification of the procedure presented by Ma and colleagues4 we developed a method to isolate CSCs from the ovarian cancer cell lines. Our protocol is advantageous as it yields more viable cells while using less toxic chemotherapeutic agents. Cells are treated with cisplatin and subsequently grown in serum-free media supplemented with growth factors (stem cell media). We isolate the resulting non-adherent sphere cells and assay them for their expression of stem cell markers. This model enables the study of CSC properties and response to drug therapy.

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		</tr>
		<tr height="101">
			<td height="101" style="height:101px;width:216px;">Primers</td>
			<td style="width:71px;">Integrated DNA Technology</td>
			<td style="width:119px;">individually designed and ordered (see protocol for sequnces)</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Anti-CD133 PE</td>
			<td style="width:71px;">Milenyl</td>
			<td>130-098-826</td>
			<td style="width:167px;">Primer/probe sets are light sensitive</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">CD117-Biotin</td>
			<td style="width:71px;">Miltenly</td>
			<td>130-098-570</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:216px;">AntiBiotin-FITC</td>
			<td style="width:71px;">Miltenly</td>
			<td>130-098-796</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P6148-1KG</td>
			<td style="width:167px;">Caution: Toxic always prepare in hood and make fresh.</td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Trypsin</td>
			<td style="width:71px;">Life Technologies</td>
			<td><a href="http://www.lifetechnologies.com/order/catalog/product/25300062">25300062</a></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">MTT (3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide)&#160;</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">25200-072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">EVOS Fl Epifluorescence and Transmitted Light Microscope</td>
			<td>Advanced Microscopy Group</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;">Biorad CFX96 C1000 System</td>
			<td style="width:71px;">Biorad</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;">Beckman Coulter FC500 Flow Cytometer&#160;</td>
			<td style="width:71px;">Beckman Coulter</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="61">
			<td height="61" style="height:61px;width:216px;">Spectramax 340PC384&#160;</td>
			<td style="width:71px;">Molecular Devices</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

enrichment for chemoresistant ovarian cancer stem cells from human cell lines

Células madre del cáncer (CSC) se definen como un subconjunto de ciclismo lento y células indiferenciadas que dividen asimétricamente para generar altamente proliferativa, invasiva, y las células tumorales quimiorresistentes. Por lo tanto, los CAC son una población de células atractivo para apuntar terapéuticamente. CSC se predice para contribuir a un número de tipos de tumores malignos, incluyendo las de la sangre, cerebro, pulmón, tracto gastrointestinal, próstata y ovario. Aislar y enriquecer una población de células tumorales de células madre cancerosas permitirá a los investigadores estudiar las propiedades, la genética, y la respuesta terapéutica de células madre cancerosas. Hemos generado un protocolo que enriquece de forma reproducible para los CSC de cáncer de ovario a partir de líneas celulares de cáncer de ovario (SKOV3 y OVCA429). Las líneas celulares se tratan con 20 M de cisplatino durante 3 días. Las células supervivientes son aislados y cultivados en un medio de células madre libre de suero que contiene citoquinas y factores de crecimiento. Se demuestra un enriquecimiento de estas células madre cancerosas purificadas mediante el análisis de las células aisladas para known marcadores de células madre Oct4, Nanog, y PROM1 (CD133) y la superficie celular expresión de CD177 y CD133. La exposición CSC incrementó chemoresistance. Este método para el aislamiento de células madre cancerosas es una herramienta útil para estudiar el papel de las células madre cancerosas en la quimiorresistencia y recidiva tumoral.

Para demostrar que se aislaron células madre cancerosas a partir de líneas celulares de cáncer de ovario epitelial utilizando el tratamiento con cisplatino, primero adquirimos imágenes de las líneas de células antes del tratamiento y después de la selección. Se utilizó microscopía de luz para capturar imágenes de adherente (no tratada) SKOV3 y OVCA429 células y SKOV3 y OVCA429 CSC (Figura 1). CSC aparecen redonda y no unido a las placas de cultivo de tejido (Figuras 1 y <str...

Watch this Scientific Journal Video about Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines at JoVE.com

Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Las células madre del cáncer (CSC) están implicados en la recidiva del tumor debido a la quimio-resistencia. Hemos optimizado un protocolo para la selección y expansión de células madre cancerosas de líneas celulares de cáncer de ovario. Mediante el tratamiento de las células con el cisplatino quimioterapia y cultivar en una célula madre promoción de los medios de comunicación que enriquecen las culturas CSC no adherentes.

El enriquecimiento de las células madre quimiorresistente cáncer ovárico de líneas celulares humanas

Cancer stem cells (CSCs) are defined as a subset of slow cycling and undifferentiated cells that divide asymmetrically to generate highly proliferative, ...

enrichment-for-chemoresistant-ovarian-cancer-stem-cells-from-human

Research

JoVE Journal

Medicine

13.4K vistas.  Indiana University School of Medicine.  Las células madre del cáncer (CSC) están implicados en la recidiva del tumor debido a la quimio-resistencia. Hemos optimizado un protocolo para la selección y expansión de células madre cancerosas de líneas celulares de cáncer de ovario. Mediante el tratamiento de las células con el cisplatino quimioterapia y cultivar en una célula madre promoción de los medios de comunicación que enriquecen las culturas CSC no adherentes. 

Video: Enrichment for Chemoresistant Ovarian Cancer Stem Cells from Human Cell Lines

Isolation of Cardiomyocyte Nuclei from Post-mortem Tissue

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events in cardiac myocytes such as proliferation and apoptosis require an accurate identification of cardiac myocyte nuclei to analyze cellular renewal in homeostasis and in pathological conditions2. Here, we provide a method to isolate cardiomyocyte nuclei from post mortem tissue by density sedimentation and immunolabeling with antibodies against pericentriolar material 1 (PCM-1) and subsequent flow cytometry sorting. This strategy allows a high throughput analysis and isolation with the advantage of working equally well on fresh tissue and frozen archival material. This makes it possible to study material already collected in biobanks. This technique is applicable and tested in a wide range of species and suitable for multiple downstream applications such as carbon-14 dating3, cell-cycle analysis4, visualization of thymidine analogues (e.g. BrdU and IdU)4, transcriptome and epigenetic analysis.

Cardiac nuclei are isolated via density sedimentation and immunolabeled with antibodies against pericentriolar material 1 (PCM-1) to identify and sort cardiomyocyte nuclei by flow cytometry.

El aislamiento de núcleos de cardiomiocitos a partir de tejidos post-mortem

Identification of cardiomyocyte nuclei has been challenging in tissue sections as most strategies rely only on cytoplasmic marker proteins1. Rare events ...

Investigación

Medicina

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Humanos neuroendocrinos líneas de células tumorales como un modelo tridimensional para el estudio de la terapia humana tumor neuroendocrino

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

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 ...

Time-lapse Imaging of Primary Preneoplastic Mammary Epithelial Cells Derived from Genetically Engineered Mouse Models of Breast Cancer

Time-lapse imaging can be used to compare behavior of cultured primary preneoplastic mammary epithelial cells derived from different genetically engineered mouse models of breast cancer. For example, time between cell divisions (cell lifetimes), apoptotic cell numbers, evolution of morphological changes, and mechanism of colony formation can be quantified and compared in cells carrying specific genetic lesions. Primary mammary epithelial cell cultures are generated from mammary glands without palpable tumor. Glands are carefully resected with clear separation from adjacent muscle, lymph nodes are removed, and single-cell suspensions of enriched mammary epithelial cells are generated by mincing mammary tissue followed by enzymatic dissociation and filtration. Single-cell suspensions are plated and placed directly under a microscope within an incubator chamber for live-cell imaging. Sixteen 650 &mu;m x 700 &mu;m fields in a 4x4 configuration from each well of a 6-well plate are imaged every 15 min for 5 days. Time-lapse images are examined directly to measure cellular behaviors that can include mechanism and frequency of cell colony formation within the first 24 hr of plating the cells (aggregation versus cell proliferation), incidence of apoptosis, and phasing of morphological changes. Single-cell tracking is used to generate cell fate maps for measurement of individual cell lifetimes and investigation of cell division patterns. Quantitative data are statistically analyzed to assess for significant differences in behavior correlated with specific genetic lesions.

Time-lapse imaging is used to assess behavior of primary preneoplastic mammary epithelial cells derived from genetically engineered mouse models of breast cancer risk to determine if there are correlations between specific behavioral parameters and distinct genetic lesions.

Time-lapse de imágenes de Primaria preneoplásicas células epiteliales mamarias derivadas de los modelos de ratones genéticamente modificados del cáncer de mama

Time-lapse  imaging can be used to compare behavior of cultured primary preneoplastic  mammary epithelial cells derived from different genetically ...

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

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 ...

Method for Obtaining Primary Ovarian Cancer Cells From Solid Specimens

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a valuable tool for understanding ovarian cancer, their use has many limitations. These include the lack of heterogeneity and the plethora of genetic alterations associated with extended in vitro passaging.&#160;Here we describe a method that allows for rapid establishment of primary ovarian cancer cells form solid clinical specimens collected at the time of surgery. The method consists of subjecting clinical specimens to enzymatic digestion for 30 min. The isolated cell suspension is allowed to grow and can be used for downstream application including drug screening. The advantage of primary ovarian cancer cell lines over established ovarian cancer cell lines is that they are representative of the original specific clinical specimens they are derived from and can be derived from different sites whether primary or metastatic ovarian cancer.

This study describes a detailed method for isolation and characterization of primary ovarian cancer cells from solid clinical specimens. Ovarian cancer clinical specimens are subjected to enzymatic digestion to obtain viable, fibroblast-free epithelial ovarian cancer (EOC) cells highly suitable for downstream applications.

Método para la obtención de las células del cáncer de ovario primarios a partir de muestras sólidas

Reliable tools for investigating ovarian cancer initiation and progression are urgently needed. While the use of ovarian cancer cell lines remains a ...

Evaluation of Stem Cell Properties in Human Ovarian Carcinoma Cells Using Multi and Single Cell-based Spheres Assays

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem cells&#8221; (CSCs) responsible for tumor initiation, maintenance and relapse following conventional chemotherapies. Identification of ovarian CSCs is therefore an important goal. A commonly used method to assess CSC potential in vitro is the spheres assay in which cells are plated under non-adherent culture conditions in serum-free medium supplemented with growth factors and sphere formation is scored after a few days. Here, we review currently available protocols for human ovarian cancer spheres assays and perform a side-by-side analysis between commonly used multi cell-based assays and a more accurate system based on single cell plating. Our results indicate that both multi cell-based as well as single cell-based spheres assays can be used to investigate sphere formation in vitro. The more laborious and expensive single cell-based assays are more suitable for functional assessment of individual cells and lead to overall more accurate results while multi cell-based assays can be strongly influenced by the density of plated cells and require titration experiments upfront. Methylcellulose supplementation to multi cell-based assays can be effectively used to reduce mechanical artifacts.

In vitro spheres assays are commonly used to identify cancer stem cells. Here we compare single with multi cell-based spheres assays. The more laborious single cell-based assays or methylcellulose supplementation give more accurate results while multi cell-based assays performed in liquid medium can be highly influenced by cell density.

Evaluación de las propiedades de células madre en células de carcinoma de ovario humano utilizando multi e individuales basados ​​en células Esferas ensayos

Years of research indicates that ovarian cancers harbor a heterogeneous mixture of cells including a subpopulation of so-called &#8220;cancer stem ...

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor initiation and/or relapse. Clarifying the mechanisms by which these tumor-initiating cells (TICs) support tumor formation and progression could lead to the development of clinically favorable therapies. Ovarian cancer is a heterogeneous and highly recurrent disease. Recent studies suggest TICs may play an important role in disease biology. We have identified culture conditions that enrich for TICs from ovarian cancer cell lines. Growing either adherent cells or non-adherent &#8216;floater&#8217; cells in a low attachment plate with serum free media in the presence of growth factors supports the propagation of ovarian cancer TICs with stem cell markers (CD133 and ALDH activity) and increased tumorigenicity without the need to physically separate the TICs from other cell types within the culture. Although the presence of floater cells is not common for all cell lines, this population of cells with innate low adherence may have high tumorigenic potential.Compared to adherent cells grown in the presence of serum, TICs readily form spheres, are significantly more tumorigenic in mice, and express putative stem cell markers. The conditions are easy to establish in a timely manner and can be used to study signaling pathways important for maintaining stem characteristics, and to identify drugs or combinations of drugs targeting TICs. The culture conditions described herein are applicable for a variety of ovarian cancer cells of epithelial origin and will be critical in providing new information about the role of TICs in tumor initiation, progression, and relapse.

In vitro Enrichment of Ovarian Cancer Tumor-initiating Cells

Tumor-initiating cells (TICs) may represent a viable therapeutic target for the treatment of ovarian cancer, a highly recurrent and fatal disease. We present a protocol for culture conditions that enrich for this highly tumorigenic population of cells.

En Enriquecimiento vitro de células iniciadoras del tumor del cáncer de ovario

Evidence suggests that small subpopulations of tumor cells maintain a unique self-renewing and differentiation capacity and may be responsible for tumor ...

Mammosphere Formation Assay from Human Breast Cancer Tissues and Cell Lines

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that self-renew while giving rise to more differentiated progeny. Understanding and targeting these cancer stem cells in breast cancer, which may possess enhanced chemo- and radio-resistance compared to the non-stem tumor bulk, has become an important research area. Markers including CD44, CD24, and ALDH activity can be assessed using fluorescence activated cell sorting (FACS) to prospectively isolate cells that display enhanced tumorigenicity when implanted into immunocompromised mice: the mammosphere assay has also become widely used for its ability to retrospectively identify sphere-forming cells that develop from single stem cell-like clones. Here we outline approaches for the appropriate culturing of mammospheres from cell lines or primary patient samples, their passaging, and calculations to estimate sphere forming efficiency (SFE). First we discuss key considerations and pitfalls in the appropriate planning and interpretation of mammosphere experiments.

Floating mammosphere assays can investigate the subset of stem-like breast cancer cells that survive in suspension conditions and show enhanced tumorigenesis when implanted into mice. This protocol provides a convenient in vitro measure of sphere-forming ability, a proxy for in vivo tumorigenesis, while facilitating analysis of the stem-associated transcriptional landscape.

Mammosphere Ensayo Formación de cáncer de mama humano tejidos y líneas celulares

Similar to healthy tissues, many blood and solid malignancies are now thought to be organised hierarchically, with a subset of stem-like cancer cells that ...