The authors would like to thank Joshua Kimani, clinical director of the research program at the University of Nairobi, for his assistance with mucosal immunology studies related to this protocol. The authors would like to acknowledge funding from CHVI grant MOP 86721.

Declaración de Ética: La ética tableros de investigación, tanto de la Universidad de Manitoba y Kenyatta Nacional Hospital / Universidad de Nairobi aprobó este estudio y el consentimiento informado por escrito se obtuvo de todos los participantes en el estudio. 1. Preparación de Medios y CMC Collection Tubes <ol><li> Preparar tampón fosfato salino (PBS) (NaCl 137.93 mM, 2,67 mM KCl, 8,1 mM Na 2 HPO 4, 1,47 mM KH 2 PO 4). Autoclave en la esterilidad. Esto se puede almacenar a 4 º C durante varios meses. </li><li> Pre-alícuota de 5 ml de PBS en 50 ml tubo Falcon (uno por cada muestra que se recogen) y se almacena a 4 º C hasta el momento de la recogida de muestras. </li><li> Etiquetar 2-4 crioviales o 1,5 ml tubos de microcentrífuga por muestra para recoger el lavado para su almacenamiento. </li><li> Preparar los medios de cultivo de células: RPMI 1640 + 1% de penicilina / estreptomicina / anfotericina B (concentración final 100 unidades / ml, 100 mg / 250 ng / ml ml y, respectively, sin FCS / FBS agregó. </li></ol> 2. Toma de muestras citocepillo Recogida de muestras cytobrush es un procedimiento no invasivo que debe ser realizada por un médico o ginecólogo entrenado. <ol><li> Recoger las células mononucleares de cuello uterino (CMC) utilizando tanto un cytobrush y un raspador de madera o plástico. Recoger CMC del participante bajo examen con espéculo mediante la inserción del rascador y que gira alrededor del orificio cervical (Figura 1). Inserte el cytobrush en el sistema operativo endocervicales, girar 360 °, e inmediatamente colocar tanto el cytobrush y el raspador en el tubo Falcon de 50 ml que contiene 5 ml de PBS. </li><li> Mantener las muestras en hielo / a 4 º C hasta su procesamiento. Para mantener la viabilidad celular, muestras de proceso dentro de las 2 horas de la recogida. </li><li> Si es posible, recoger muestras de sangre coincidentes en la tapa verde de tubos vacutainer de heparina para células mononucleares de sangre periférica (PBMC) de aislamiento para servir como un comparador / de control para el análisis de CMC. </li&gt; </ol> 3. Aislamiento de CMC Lleve a cabo el procesamiento de muestras en un laboratorio de bioseguridad de nivel 2, en un gabinete de bioseguridad estéril con guantes dobles. <ol><li> Durante la recolección, las muestras de CMC pueden contaminarse con sangre. Excluir las muestras con contaminación de la sangre visible desde el estudio con el fin de evitar la inclusión de confusión de PBMC en la muestra final. Debido al bajo número de linfocitos aislados de muestras de CMC, contaminación de la sangre menor de edad puede fácilmente abrumar el componente de linfocitos CMC. </li><li> Tubos Falcon Vortex que contienen tanto el cytobrush y rascador durante 45 segundos para disociar las células de los cepillos. Las muestras pueden ser espumosa durante este proceso; esto es normal. </li><li> Utilice la cytobrush para raspar cualquier material restante de la rasqueta, y desechar el rascador en una solución de cloro. Para recoger las células permanezcan unidas al cepillo / raspador, use un guante fresco para exprimir el rascador entre el pulgar y el índice.Deslízate por el pincel, permitiendo que las células y PBS que se recojan en el mismo tubo de 50 ml. Deseche el cytobrush en una solución de cloro, y deseche el guante. IMPORTANTE: Utilice un guante nuevo para cada muestra. </li><li> Montar un 100 m de células colador de nylon a un tubo Falcon de 50 ml fresco. Usando una pipeta de transferencia, recoger la suspensión de células de PBS desde el tubo de muestra y filtrado a través del filtro en el tubo fresco. </li><li> Añadir 5 ml de medio RPMI 1640 al tubo de muestra original. Utilizando la pipeta de transferencia, lavar los lados del tubo de muestra con el medio RPMI, y transferir a través del filtro para el nuevo tubo de recogida. Lavar la parte inferior del filtro de nylon con el medio RPMI así. </li><li> Centrifugar las muestras durante 10 min a 514 x g. Es crucial para salir de la freno de la centrífuga fuera durante este paso, con el fin de evitar el desplazamiento de la pastilla de CMC. </li><li> Retire con cuidado el sobrenadante del tubo, teniendo cuidado de no perturbar el sedimento. Tamaño de pellets será muy variable; en lome muestras, la pastilla es bastante grande debido a la presencia de grandes números de células epiteliales, mientras que en otros casos, el sedimento es apenas visible y altamente transparente. </li><li> Resuspender el precipitado por una ligera agitación del tubo Falcon. Añadir 5 ml de PBS para lavar la muestra. </li><li> Se centrifuga de nuevo durante 10 min a 514 xg sin freno de la centrífuga. </li><li> Elimine con cuidado el sobrenadante y volver a suspender el sedimento como anteriormente. Las células están listas, ya sea para la crioconservación (usando un protocolo de criopreservación de PBMC), la estimulación o la citometría de flujo fenotipificación. </li></ol> 4. CMC tinción de la superficie y Citometría de Flujo <ol><li> Resuspender el sedimento de la etapa 3.10 en 100 l de solución de bloqueo y transferir ya sea en una placa de 96 pocillos o un tubo de FACS. La solución de bloqueo (para bloquear los receptores Fc gamma) receta es la siguiente: IgG de ratón 1,8 l (concentración final 0,2 g / l), 5 l de FBS y 93,2 l de FACS de lavado (PBS 2% de FBS).La tinción se puede realizar en una placa de 96 pocillos si los tamaños de pellets son lo suficientemente pequeños, pero puede necesitar ser realizado en tubos de FACS si gránulos son rutinariamente grande. Es importante para titular anticuerpos en consecuencia. </li><li> Bloquee las muestras durante 10 minutos en hielo / a 4 ° C. </li><li> Lavar las células con 100 l de FACS de lavado (PBS 2% de FBS) en placas de 96 pocillos, o 500 l en tubos de FACS. Se centrifuga a 600 × g durante 10 min a 4 ° C con el freno de baja (o desactivar, si un nivel bajo de freno no está disponible). </li><li> Eliminar el sobrenadante y volver a suspender el sedimento celular por agitación. Añadir Stain viabilidad (Live colorante de viabilidad fixable Dead). Preparar colorante de viabilidad de acuerdo con las instrucciones del fabricante. Es posible que haya que valora para su uso en cada configuración de laboratorio / citómetro. Incubar durante 30 min en la oscuridad a 4 ° C. </li><li> Lavar las células 100 μl/500 l de PBS (para tubos de placa / FACS de 96 pocillos). Se centrifuga a 600 × g durante 10 min con baja / no freno. Retire supernatant y se resuspenden las células. </li><li> Se incuban las células con el cóctel de anticuerpos de marcadores de superficie, en un volumen total de 100 l. IMPORTANTE: Optimizar y valorar cada una de citometría de flujo de la comisión antes de probar las manchas. Se incuban las células durante 30 minutos en la oscuridad a 4 ° C. </li><li> Repita el paso 4.5. si la tinción en una placa de 96 pocillos, las células de transferencia a un tubo de FACS y se diluye hasta un volumen final de 750 l de FACS de lavado que contiene 1% de paraformaldehído (PFA) (o CytoFix, se diluyó 1 en 4). Proceder a la adquisición de datos en un citómetro de flujo. </li></ol> 5. Recogida y preparación de Cervical Vaginal Lavado (CVL) <ol><li> Antes de citocepillo de muestreo, utilizar una jeringa para lavar la endocérvix con 2 ml de PBS estéril y aspirar el lavado del fórnix posterior. </li><li> Recoger la muestra de lavado aspirado en un tubo Falcon de 15 ml y mantener en hielo / a 4 º C hasta su procesamiento. </li><li> Centrifugar la muestra a 400 xg durante 7 minutos para eliminar los desechos celulares. </li><li> Collect el sobrenadante, alícuota de la muestra en 1 ml o 500 ml de alícuotas y se almacena a -70 ° C. Las alícuotas se pueden descongelar para ELISA o análisis de bolas de serie de proteínas CVL, pero evitar los ciclos de congelación / descongelación. </li><li> Si se desea, resuspender el sedimento de restos celulares en el ARN Más tarde para medir la expresión de ARN siguiendo el protocolo del fabricante. </li></ol> 6. Adquisición de Datos <ol><li> Prepare un conjunto de tubos de compensación que coincide con el panel de anticuerpos. Ejecutar los tubos de compensación para ajustar la superposición espectral. Ejecute las muestras de ningún flujo multicolor citómetro de que está configurado para los anticuerpos conjugados con fluorocromos utilizados en el panel. </li><li> Adquirir una muestra PBMC primero para ajustar la dispersión hacia adelante (FSC) y la dispersión lateral (SSC) de tensión a fin de detectar la población de linfocitos. Trate de mantenerlos a 100 en cada eje en una escala lineal. Ejecución de un control de PBMC hará que sea mucho más fácil para encontrar la población de linfocitos CMC. </li><li> El umbral de FSC para las muestras de CMC puede ser necesario un aumento con respecto a las muestras de PBMC debido a manchar el eje SSC a bajos valores de FSC. </li><li> Agite cada tubo antes de adquirir. Adquirir la totalidad del tubo para maximizar el número de eventos puerta de linfocitos recogidos. Vigilar la máquina con cuidado para evitar obstrucciones. </li><li> Exportar los datos a un programa de análisis de citometría de flujo tal como FlowJo. </li></ol> 7. Estrategia Gating <ol><li> Seleccione dispersión lateral delantero alto (FSC-H) / área de dispersión lateral frontal (FSC-A) para determinar la población singlete. </li><li> Seleccione Hora frente marcador fluorescente (como ejemplo FITC) para controlar por la calidad de la adquisición. Cambio en la tasa de flujo puede introducir artefactos en los datos. Excluir cualquier área que muestra la discrepancia en el caudal. </li><li> Identificar la población de linfocitos en SSC-A/FSC-A parcela. Utilice un control PBMC para facilitar la identificación. Tenga en cuenta que la población de linfocitos CMC podría no ser tan claro como el control de PBMC. &lt;/ Li&gt; <li> Puerta en SSC-A/viability tinte para excluir las células muertas de células vivas. Las células muertas pueden incorporar de forma no específica de los anticuerpos, que introducirá los artefactos. </li><li> Puerta en la SSC-A/CD3 para identificar la población de células T. Desde esta puerta, identificar las poblaciones CD4 + y CD8 +. </li></ol>

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The authors have nothing to disclose.

Dadas las grandes lagunas de conocimiento con respecto a la inmunidad en el tracto genital femenino (FGT), el análisis fenotípico de los CMC puede proporcionar una amplia gama de puntos de vista en varias poblaciones de linfocitos en el cuello uterino. Junto con el análisis proteómico y mediciones de carga viral en el lavado de cuello de útero, la inmunidad a las infecciones de transmisión sexual (ITS) s y otros patógenos puede ser diseccionado en varias poblaciones. Considera...

La mayoría de las nuevas infecciones por el VIH en todo el mundo surgen a través de la transmisión heterosexual, las mujeres representan el 47% de las nuevas infecciones en 2011 (ONUSIDA 1). Entender el tracto genital femenino (FGT), uno de los principales portales de entrada para el VIH y otros agentes patógenos de transmisión sexual, es de gran importancia en el camino hacia la búsqueda de estrategias eficaces para prevenir la infección. Las respuestas inmunes en la mucosa genital son claramente únicas y difieren de los medidos en la sangre periférica 2. Sin embargo, los conocimientos actuales de la dinámica del sistema inmune en el FGT se limita, en el mejor. Hasta la fecha, los estudios sobre el medio ambiente inmune de la mucosa se ​​han centrado en gran medida en los tejidos linfoides asociados al intestino (GALT), donde se ha convertido en claro que los primeros eventos en los tejidos de las mucosas después de la infección tienen un fuerte impacto en la progresión de la enfermedad posterior 3,4. Recogida de muestras de la mucosa genital representa un gran desafío y es al menos parcialmente responsable de la lack de comprensión de la inmunología de la FGT. Resolviendo el rompecabezas de la dinámica inmunitaria entre el huésped y el patógeno en el contexto del entorno distinto que es el FGT requiere métodos eficaces para la recogida y análisis de muestras de esta localidad. El FGT se divide en dos secciones: el tracto reproductivo superior que incluye las trompas de Falopio, endometrio y endocérvix, y el tracto inferior que contiene el exocérvix y la vagina (revisado por Kaushic et al 5). Aún no está claro lo que la contribución relativa de estos diferentes sitios es a la infección por el VIH, pero se cree que ambos sitios podrían contribuir a la entrada del VIH 6. Células T representan el 40-50% de los leucocitos en los tractos reproductivos superior e inferior, mientras que los macrófagos comprenden aproximadamente el 10% (revisado en Rodríguez-García et al 2). Las células T pueden ser detectados en la vagina, el cuello uterino, y endometrio. Los macrófagos son más fuertemente localizeta en el endometrio y el tejido conectivo del miometrio que el cuello del útero, a pesar de que se pueden detectar en ambos tejidos. Por último, las células dendríticas plasmacitoides (PDC) y células de Langerhans también pueden ser detectados en los tejidos de TGC. El fenotipo y proporciones de poblaciones inmunes y su susceptibilidad a la infección por el VIH pueden variar importante de acuerdo a los ciclos hormonales, el uso de anticonceptivos hormonales, la vaginosis bacteriana o actividades sexuales 5,7-9. Diversos métodos se han desarrollado para estudiar las poblaciones inmunes y el medio ambiente de la FGT. Biopsia cervical, cepillados cervicales y lavados cervicovaginales (CVL) 10-12 son los más utilizados en toda la literatura. Colección CVL por PBS lavado es el método más simple y permite el estudio de proteínas inmunomoduladoras, pero los resultados en rendimiento extremadamente bajo de células, y por lo tanto no es adecuado para el estudio de las poblaciones de células inmunes del TGC 13. Muestras CVL son, por otra parte, VEry útil para evaluar el entorno inmunológico del TGC mediante la medición de la expresión de diversas citoquinas, quimioquinas o factores antimicrobianos utilizando métodos tales como ELISA, de bolas de serie de citoquinas 14 o espectrometría de masas 15,16. Caracterización de las frecuencias de células inmunes, fenotipos y funciones se puede lograr mediante la recopilación de las células mononucleares de cuello uterino (CMC) por citocepillo cervical o mediante la toma de muestras de biopsia cervical. Toma de muestras de biopsia de cuello uterino es un método invasivo que aumenta el malestar y el riesgo de sangrado y tarda de 2 a 11 días en sanar después del procedimiento, dependiendo del estado inmunológico de la mujer 12. Por otro lado, cepillados cervicales, a pesar de la menor rendimiento de las células recogidas, es un método menos invasivo y más conveniente para recoger las células inmunes de la TGC. Ambos métodos pueden alcanzar el mismo rendimiento de leucocitos CD45 +, pero dos cepillados cervicales secuenciales son necesarias para obtener la misma cantidad de células contenidas in una biopsia 13. Sin embargo, el muestreo citocepillo todavía proporciona un número aceptable de células (células CD45 + sobre 5000 / citocepillo) para obtener más fenotipificación ex vivo mediante citometría de flujo 14. Además, la caracterización funcional puede llevarse a cabo en estas muestras, como se han realizado la estimulación y citometría de flujo intracelular o qPCR utilizando CMC cytobrush derivados para identificar respuestas inmunes específicas para el VIH 17 o polarización de las células Th. 18. La expansión de la población de células T también puede facilitar los estudios funcionales con CMC 19. Es importante tener en cuenta que las biopsias y cepillados muestrean porciones distintas de la TGC. Las biopsias se derivan de la parte superior del epitelio y el estroma del exocérvix 12,13, mientras cepillados cervicales muestrean del orificio cervical, la recolección de células derivadas del epitelio del endocérvix y, presumiblemente, la zona de transformación. Por lo tanto, las muestras citocepillo muestrean una reregión compone de una sola capa de epitelio cilíndrico, mientras que las biopsias, incluyen una región de la línea por un epitelio escamoso estratificado 5. Como resultado, la naturaleza de las poblaciones de leucocitos recogidos por biopsia cervical y citocepillo difiere. Las biopsias recogen una mayor proporción de células T CD3 +, mientras que cepillados resultan en la colección de una mayor proporción de monocitos CD14 + / macrófagos 13. El estudio de la inmunología de la FGT ha habido un interés por muchos años 20-22 y hemos acumulado una gran experiencia con el estudio de los CMC cytobrush derivados. Nuestros estudios se centran principalmente en el estudio de la infección por el VIH, no infectados y expuestos al VIH seronegativos (HESN) trabajadoras sexuales de Nairobi, Kenia. El VIH se replica preferentemente en células T activadas 23 e inferior del número de células activadas que pueden ser dirigidos por el VIH en el TGC podrían contribuir a la protección contra la adquisición del VIH. De acuerdo con esta hipótesis, varios studies han descrito la activación inmune más baja entre los trabajadores del sexo HESN que están altamente expuestos al VIH aún permanecen infectados 24,25 y este fenotipo quiescente se observa también en el TGC 14. Aquí se describe la metodología para la elaboración y evaluación de la activación de células T en muestras de CMC derivados de cepillados cervicales por ex vivo citometría de flujo.

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collection, isolation, and flow cytometric analysis of human endocervical samples

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female genital tract (FGT). Because heterosexual transmission now represents the dominant mechanism of HIV transmission, and given the continual spread of sexually transmitted infections (STIs), it is critical to understand the interplay between host and pathogen at the genital mucosa. The substantial gaps in knowledge around FGT immunity are partially due to the difficulty in successfully collecting and processing mucosal samples. In order to facilitate studies with sufficient sample size, collection techniques must be minimally invasive and efficient. To this end, a protocol for the collection of cervical cytobrush samples and subsequent isolation of cervical mononuclear cells (CMC) has been optimized. Using ex vivo flow cytometry-based immunophenotyping, it is possible to accurately and reliably quantify CMC lymphocyte/monocyte population frequencies and phenotypes. This technique can be coupled with the collection of cervical-vaginal lavage (CVL), which contains soluble immune mediators including cytokines, chemokines and anti-proteases, all of which can be used to determine the anti- or pro-inflammatory environment in the vagina.

Citometría de flujo multiparamétrica es una herramienta poderosa para diseccionar los fenotipos y las funciones de los subconjuntos de células en los tejidos previamente sin caracterizar. Análisis de muestras de CMC puede dar información tanto de linfocitos y monocitos poblaciones con estrategias apropiadas de compuerta. Una estrategia de activación periódica CMC representante, en comparación con un perfil de PBMC emparejado, se muestra en la Figura 2. El FSC-A frent...

Watch this Scientific Journal Video about Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples at JoVE.com

Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays.

Colección, Aislamiento, y citometría de flujo análisis de las muestras endocervicales Humano

Despite the public health importance of mucosal pathogens (including HIV), relatively little is known about mucosal immunity, particularly at the female ...

collection-isolation-flow-cytometric-analysis-human-endocervical

Research

JoVE Journal

Medicine

19.0K vistas.  University of Manitoba. The use of cytobrush sampling to collect lymphocytes and monocytes from the endocervix is a minimally invasive technique that provides samples for analysis of female genital tract immunity. In this protocol, we describe the collection of cytobrush samples and immune cell isolation for flow cytometry assays. 

Video: Collection, Isolation, and Flow Cytometric Analysis of Human Endocervical Samples

Isolation of Human Islets from Partially Pancreatectomized Patients

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 diabetic islets from organ donors2. Here we share our protocol for isolating islets from human pancreatic tissue obtained from type 2 diabetic and non-diabetic patients who have undergone partial pancreatectomy due to different pancreatic diseases (benign or malignant pancreatic tumors, chronic pancreatitis, and common bile duct or duodenal tumors). All patients involved gave their consent to this study, which had also been approved by the local ethics committee. The surgical specimens were immediately delivered to the pathologist who selected soft and healthy appearing pancreatic tissue for islet isolation, retaining the damaged tissue for diagnostic purposes. We found that to isolate more than 1,000 islets, we had to begin with at least 2 g of pancreatic tissue. Also essential to our protocol was to visibly distend the tissue when injecting the enzyme-containing media and subsequently mince it to aid digestion by increasing the surface area.
To extend the applicability of our protocol to include the occasional case in which a large amount (&gt;15g) of human pancreatic tissue is available , we used a Ricordi chamber (50 ml) to digest the tissue. During digestion, we manually shook the Ricordi chamber3 at an intensity that varied by specimen according to its level of tissue fibrosis. A discontinous Ficoll gradient was then used to separate the islets from acinar tissue. We noted that the tissue pellet should be small enough to be homogenously resuspended in Ficoll medium with a density of 1.125 g/ml. After isolation, we cultured the islets under stress free conditions (no shaking or rotation) with 5% CO2 at 37 &deg;C for at least 48 h in order to facilitate their functional recovery. Widespread application of our protocol and its future improvement could enable the timely harvesting of large quantities of human islets from diabetic and clinically matched non-diabetic subjects, greatly advancing type 2 diabetes research.

The supply of type 2 diabetic islets for research is insufficient. Here we share our protocol for isolating islets from patients undergoing partial pancreatectomy. This approach represents a unique venue for obtaining islets from type 2 diabetic and clinically matched non-diabetic subjects in adequate numbers for basic and clinical studies.

El aislamiento de islotes humanos de pacientes parcialmente Pancreatectomized

Investigations into the pathogenesis of type 2 diabetes and islets of Langerhans malfunction 1 have been hampered by the limited availability of type 2 ...

Investigación

Medicina

Collection Protocol for Human Pancreas

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation of pancreas recovered from cadaveric organ donors. The pancreas is divided into 3 main regions (head, body, tail) followed by serial transverse sections throughout the medial to lateral axis. Alternating sections are used for fixed paraffin and fresh frozen blocks and remnant samples are minced for snap frozen sample preparations, either with or without RNAse inhibitors, for DNA, RNA, or protein isolation. The overall goal of the pancreas dissection procedure is to sample the entire pancreas while maintaining anatomical orientation.
Endocrine cell heterogeneity in terms of islet composition, size, and numbers is reported for human islets compared to rodent islets 1. The majority of human islets from the pancreas head, body and tail regions are composed of insulin-containing &beta;-cells followed by lower proportions of glucagon-containing &alpha;-cells and somatostatin-containing &delta;-cells. Pancreatic polypeptide-containing PP cells and ghrelin-containing epsilon cells are also present but in small numbers. In contrast, the uncinate region contains islets that are primarily composed of pancreatic polypeptide-containing PP cells 2. These regional islet variations arise from developmental differences. The pancreas develops from the ventral and dorsal pancreatic buds in the foregut and after rotation of the stomach and duodenum, the ventral lobe moves and fuses with the dorsal 3. The ventral lobe forms the posterior portion of the head including the uncinate process while the dorsal lobe gives rise to the rest of the organ. Regional pancreatic variation is also reported with the tail region having higher islet density compared to other regions and the dorsal lobe-derived components undergoing selective atrophy in type 1 diabetes4,5.
Additional organs and tissues are often recovered from the organ donors and include pancreatic lymph nodes, spleen and non-pancreatic lymph nodes. These samples are recovered with similar formats as for the pancreas with the addition of isolation of cryopreserved cells. When the proximal duodenum is included with the pancreas, duodenal mucosa may be collected for paraffin and frozen blocks and minced snap frozen preparations.

This video demonstrates a dissection procedure for processing human pancreas into multiple storage formats. Anatomical orientation is maintained throughout the pancreatic regions to allow definition of regional islet composition and density.

Colección Protocolo de páncreas humano

This dissection and sampling procedure was developed for the Network for Pancreatic Organ Donors with Diabetes (nPOD) program to standardize preparation ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

El aislamiento, la cultura, y de imágenes de Human Fetal pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Isolation and Functional Characterization of Human Ventricular Cardiomyocytes from Fresh Surgical Samples

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and membrane ion currents. Those changes are likely&#160;to be responsible for the increased arrhythmogenic risk and the contractile alterations leading to systolic and diastolic dysfunction in cardiac patients. However, most information on the alterations of myocyte function in cardiac diseases has come from animal models.
Here we describe and validate a protocol to isolate viable myocytes from small surgical samples of ventricular myocardium from patients undergoing cardiac surgery operations. The protocol is described in detail. Electrophysiological and intracellular calcium measurements are reported to demonstrate the feasibility of a number of single cell measurements in human ventricular cardiomyocytes obtained with this method.
The protocol reported here can be useful for future investigations of the cellular and molecular basis of functional alterations of the human heart in the presence of different cardiac diseases. Further, this method can be used to identify novel therapeutic targets at cellular level and to test the effectiveness of new compounds on human cardiomyocytes, with direct translational value.

Current knowledge on the cellular basis of cardiac diseases mostly relies on studies on animal models. Here we describe and validate a novel method to obtain single viable cardiomyocytes from small surgical samples of human ventricular myocardium. Human ventricular myocytes can be used for electrophysiological studies and drug testing.

Aislamiento y caracterización funcional de Human ventricular cardiomiocitos a partir de muestras quirúrgicas recientes

Cardiomyocytes from diseased hearts are subjected to complex remodeling processes involving changes in cell structure, excitation contraction coupling and ...

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 ...

Rapid Fractionation and Isolation of Whole Blood Components in Samples Obtained from a Community-based Setting

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both with and without preexisting conditions. Rapid processing of these samples is essential to avoid degradation of key cellular components. Included here are methods for simultaneous peripheral blood mononuclear cell (PBMC), DNA, RNA and serum isolation from a single blood draw performed in the homes of consenting participants across a metropolitan area, with processing initiated within 2 hr of collection. We have used these techniques to process over 1,600 blood specimens yielding consistent, high quality material, which has subsequently been used in successful DNA methylation, genotyping, gene expression and flow cytometry analyses. Some of the methods employed are standard; however, when combined in the described manner, they enable efficient processing of samples from participants of population- and/or community-based studies who would not normally be evaluated in a clinical setting. Therefore, this protocol has the potential to obtain samples (and subsequently data) that are more representative of the general population.

We outline a methodology for the processing of whole blood to obtain a variety of components for further analysis. We have optimized a streamlined protocol that enables rapid, high-throughput simultaneous processing of whole blood samples in a non-clinical setting.

Fraccionamiento rápida y aislamiento de componentes de sangre total en las muestras obtenidas a partir de una configuración basada en la comunidad

Collection and processing of whole blood samples in a non-clinical setting offers a unique opportunity to evaluate community-dwelling individuals both ...

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic uses.
Exosomes have recently been shown to contain microRNAs (miRs). MiRs themselves have arisen as valuable biomarkers for diagnostic purposes. As specimen collection in clinics and hospitals is quite variable, miRNA isolation from whole bile varies substantially. To achieve robust, accurate and reproducible miRNA profiles from collected bile samples in a simple manner required the development of a high-quality protocol to isolate and characterize exosomes from bile. The method requires several centrifugations and a filtration step with a final ultracentrifugation step to pellet the isolated exosomes. Electron microscopy, Western blots, flow cytometry and multi-parameter nanoparticle optical analysis, where available, are crucial characterization steps to validate the quality of the exosomes. For the isolation of miRNA from these exosomes, spiking the lysate with a non-specific, synthetic miRNA from a species like Caenorhabditis elegans, i.e., Cel-miR-39, is important for normalization of RNA extraction efficiency. The isolation of exosome from bile fluid following this method allows the successful miRNA profiling from bile samples stored for several years at -80 &#176;C.

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

El aislamiento y la generación de perfiles de exosomas de la bilis humana que contiene microRNA

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

Simultaneous Flow Cytometric Characterization of Multiple Cell Types Retrieved from Mouse Brain/Spinal Cord Through Different Homogenization Methods

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central nervous system (CNS). However, further optimization of these technologies would benefit from methods allowing rapid and streamline determination of the extent of CNS and cell-specific targeting upon administration of viral vectors or nanoparticles in the body. Here, we present a protocol that takes advantage of the high throughput and multiplexing capabilities of flow cytometry to allow a straightforward quantification of different cell subtypes isolated from mouse brain or spinal cord, namely microglia/macrophages, lymphocytes, astrocytes, oligodendrocytes, neurons and endothelial cells. We apply this approach to highlight critical differences between two tissue homogenization methods in terms of cell yield, viability and composition. This could instruct the user to choose the best method depending on the cell type(s) of interest and the specific application. This method is not suited for analysis of anatomical distribution, since the tissue is homogenized to generate a single-cell suspension. However, it allows to work with viable cells and it can be combined with cell-sorting, opening the way for several applications that could expand the repertoire of tools in the hands of the neuroscientist, ranging from establishment of primary cultures derived from pure cell populations, to gene-expression analyses and biochemical or functional assays on well-defined cell subtypes in the context of neurodegenerative diseases, upon pharmacological treatment or gene therapy.

We present a flow cytometry method to identify simultaneously different cell types retrieved from mouse brain or spinal cord. This method could be exploited to isolate or characterize pure cell populations in neurodegenerative diseases or to quantify the extent of cell targeting upon in vivo administration of viral vectors or nanoparticles.

Caracterización citométrica de flujo simultáneo de múltiples tipos de células recuperadas del cerebro del ratón/cordón espinal a través de diferentes métodos de homogeneización

Recent advances in viral vector and nanomaterial sciences have opened the way for new cutting-edge approaches to investigate or manipulate the central ...

Hemocompatibility Testing of Blood-Contacting Implants in a Flow Loop Model Mimicking Human Blood Flow

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body&#39;s circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 &#176;C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (&#946;-thromboglobulin [&#946;-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Pruebas de hemocompatibilidad de implantes que contactan con la sangre en un modelo de bucle de flujo que imita el flujo sanguíneo humano

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the ...

Validated LC-MS/MS Panel for Quantifying 11 Drug-Resistant TB Medications in Small Hair Samples

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. Plasma drug levels are the current gold standard assessment, but require phlebotomy and a cold chain, and capture only very recent adherence. Our method uses hair, a matrix that is easily collected and reflective of long-term adherence, to test for 11 anti-TB medications. Previous work by our group shows that antiretroviral drug levels in hair are associated with HIV outcomes. Our method for DR-TB drugs uses 2 mg of hair (3 cm proximal to the root), which is pulverized and extracted in methanol. Samples are analyzed with a single LC-MS/MS method, quantifying 11 drugs in a 16 min run. Lower limits of quantification (LLOQs) for the 11 drugs range from 0.01 ng/mg to 1 ng/mg. Drug presence is confirmed by comparing ratios of two mass spectrometry transitions. Samples are quantified using the area ratio of the drug to the deuterated, 15N-, or 13C-labeled drug isotopologue. We used a calibration curve ranging from 0.001-100 ng/mg. Application of the method to a convenience sample of hair samples collected from DR-TB patients on directly observed therapy (DOT) indicated drug levels in hair within the linear dynamic range of nine of the eleven drugs (isoniazid, pyrazinamide, ethambutol, linezolid, levofloxacin, moxifloxacin, clofazimine, bedaquiline, pretomanid). No patient was on prothionamide, and the measured levels for ethionamide were close to its LLOQ (with further work instead examining the suitability of ethionamide&#8217;s metabolite for monitoring exposure). In summary, we describe the development of a multi-analyte panel for DR-TB drugs in hair as a technique for therapeutic drug monitoring during drug-resistant TB treatment.

Current methods of analyzing patients&#8217; adherence to complex drug resistant-tuberculosis (DR-TB) regimens can be inaccurate and resource-intensive. Our method analyzes hair, an easily collected and stored matrix, for concentrations of 11 DR-TB medications. Using LC-MS/MS, we can determine sub-nanogram drug levels that can be utilized to better understand drug adherence.

Panel LC-MS/MS validado para cuantificar 11 medicamentos resistentes a la tuberculosis en muestras de pelo pequeño

Drug resistant-tuberculosis (DR-TB) is a growing public health threat, and assessment of therapeutic drug levels may have important clinical benefits. ...