Damos las gracias a Lorna Renshaw (Unidad de Mama de Edimburgo) por su inestimable ayuda en la obtención de muestras clínicas. El uso de material tumoral fue aprobado por el Comité Ético de Investigación Lothian (08/S1101/41) y obtuvo bajo los auspicios de Medicina Experimental del Cáncer Programa de Centros (Edimburgo). Este trabajo es apoyado por el Consejo Escocés de Financiación. ADL es una señorita Margaret Butters Reekie compañeros de confianza clínica.

1. La extracción de colágeno de la cola de rata <ol><li> Lugar fresco congelado colas de ratas en el 70% de etanol. </li><li> Deglove la piel que recubre los tendones con un bisturí la exposición. </li><li> Franja de los tendones de distancia de la cola y el lugar en el 70% de etanol para esterilizar. </li><li> Pesar los tendones recogida y traslado al ácido acético (tendón de 1g a 250 ml de ácido acético 0,5 M). Mezclar durante 48 horas de grabación a 4 ° C. </li><li> Centrifugar la mezcla de ácido tendón / acético a 10.000 g durante 30 minutos y desechar el sedimento. </li><li> Añadir un volumen igual de 10% (w / v) NaCl al sobrenadante y dejar la mezcla reposar durante la noche a 4 ° C. </li><li> Recoger la capa rica en colágeno, inferior insoluble y centrifugado para 30 minutos más de 10.000 g. </li><li> Resuspender el material rico en colágeno en ácido acético 0,25 M a 4 ° C y dializa contra 1:1000 ácido acético a 4 ° C durante 3 días, cambiando el tampón de diálisis dos veces al día. </li><li> Además esterilizar la solución de colágeno por centrifugación (20.000 g durante 2 h) y se almacenan a 4 ° C. </li><li> Diluir colágeno según sea necesario con la adición de ácido acético 1:1000 estéril a una concentración de 1mg/ml de valores. </li></ol> 2. 3D Creación de ensayo El ensayo 3D se basa en un ensayo de línea celular, publicó anteriormente con el 2. <ol><li> Múltiples biopsias centrales se recolectan de consentir los pacientes en el momento de la resección curativa quirúrgica para el cáncer de mama invasivo. </li><li> A simple vista, se dividen los núcleos con un bisturí en 1 mm 3 fragmentos. Recorte y deseche la grasa macroscópicamente diferentes. </li><li> Sumerja los fragmentos en los medios de comunicación MEGM completa. El tejido se pueden almacenar en este estado durante un máximo de 1 hora. </li><li> Para crear el gel de colágeno, la mezcla de hielo de colágeno de cola de rata 1mg/ml, 1:1000 ácido acético filtrada, 10x DMEM/F12 y NaOH 0.22M en una proporción de 3:5:1:1 para crear una concentración de colágeno final de 0,3 mg / ml. </li><li> Inyectar 1,2 ml de la mezcla de colágeno en los distintos pozos de una placa de 24 pocillos y el lugar en una incubadora a 37 ° C durante 10 minutos para iniciar la gelificación. </li><li> Después de 10 minutos, retire el plato y 24 de la incubadora. Coloque los trozos de tumor en el colágeno de gelificación y suavemente empuje hacia el centro del gel usando una 10μl punta de la pipeta. Devolver la placa y 24 a la incubadora durante 1 hora. </li><li> Para los tumores ER +, suplemento MEGM los medios de comunicación completa con β-estradiol a una concentración de 3.2x10 -10 M. Una vez que los ensayos de gel tienen gel durante 1 hora, introducir con cuidado 1,2 ml de los medios de comunicación complementado en cada pozo. El final de β-estradiol concentración en el ensayo se equilibriate a 1.6x10 -10 M para recapitular los niveles fisiológicos de estrógeno que se encuentra en el tejido mamario 3. </li><li> Ejecutar una punta de pipeta 10μl alrededor del borde interior de cada bien, la liberación de cada gel circunferencialmente, permitiendo así que cada gel a flotar libremente en los medios de comunicación. Devolver la placa y 24 a la incubadora. </li><li> Intercambio complementado los medios de comunicación dos veces por semana hasta la finalización del experimento. Terminar el experimento mediante la eliminación de los medios de comunicación y la adición de un fijador de formol. </li></ol> 3. La tinción de anticuerpos del ensayo en la preparación de la tomografía óptica de proyección Hemos adoptado un método previamente publicado por 4 todo tinción de las muestras. <ol><li> Lávese las muestras fijadas en formol durante 30 minutos en PBS. Deshidratar los geles paso a paso en metanol diluido en PBS que contenía 0,05% de Tween 20 (0,05% PBST): 33%, 66% y el 100% de ≥ 15 minutos en cada paso. </li><li> Incubar el tejido recién preparada MeOH: DMSO: 30% H 2 O 2 en una proporción de 02:01:03 (15% v / v de H 2 O 2) a temperatura ambiente durante 24 horas para saciar la autofluorescencia. </li><li> Lávese las muestras dos veces durante 30 minutos en metanol. Congelar las muestras a -80 ° C cinco veces por lo menos 1 hora cada vez y de nuevo a temperatura ambiente (TA) para asegurarse de que los antígenos en las partes más profundas de los tejidos se vuelven accesibles. </li><li> Rehidratar los tejidos de nuevo a 0,05% PBST con metanol como disolvente (33%, 66% y 100% para 15 minutos en cada paso). </li><li> Bloquear el tejido en solución de bloqueo (10% de suero en el 0,05% PBST) para las 24 horas. </li><li> Incubar el tejido con el anticuerpo primario para identificar el contenido del epitelio, de conejo anti-citoqueratina a una concentración de 1:500 diluido en 1,5 ml de solución de bloqueo con 5% de DMSO durante 48 horas de grabación. </li><li> Lávese las muestras de cuatro veces con PBST 0,05% durante 30 minutos cada uno. </li><li> Incubar el tejido con anticuerpos fluorescentes secundaria (1:100 de cabra anti-conejo Alexa555), diluida en solución de 1,5 ml de bloqueo que contienen un 5% de DMSO durante 48 horas de grabación. </li><li> Lavado de las muestras una vez más con PBST 0,05% cuatro veces durante 30 minutos cada uno. </li></ol> 4. Tomografía óptica de proyección Tomografía por proyección óptica es una técnica de microscopía desarrolladas previamente para producir imágenes de alta resolución en 3D de las muestras biológicas fluorescentes con un espesor de hasta 15 milímetros 5. <ol><li> Montar el sgel sostenido de bajo punto de fusión al 1% de agarosa y se deshidratan en el 100% de metanol durante 24 horas. Clara muestra de la solución en Babb (1:2 alcohol bencílico, benzoato de bencilo) para 48 horas de grabación y escanear con el escáner OPT 3001M (Bioptonics). </li><li> Tanto el objetivo como la autofluorescencia se capturaron con el conjunto de filtros Cy3 (excitador 545nm/30 nm, emisor 610/75 nm) y GFP1 conjunto de filtros (excitación 425nm/40 nm, emisor LP475 nm), respectivamente. </li></ol> 5. La reconstrucción y cuantificación 3D <ol><li> El conjunto de proyecciones angular adquirida por OPT puede ser reconstruido utilizando NRecon software (SkyScan) para crear una serie de cortes de sección transversal a través de la invasión de ensayo. Este software está disponible para descargar de forma gratuita en <a href="http://www.skyscan.be/products/downloads.htm">http://www.skyscan.be/products/downloads.htm</a> . </li><li> Analizar los datos reconstruidos utilizando el software Volocity (PerkinElmer). Software de demostración gratuita está disponible para su descarga en <a href="http://www.cellularimaging.com/products/Volocity/demo/">http://www.cellularimaging.com/products/Volocity/demo/</a> . </li><li> Con el fin de cuantificar el volumen epiteliales mediante tinción con citoqueratina, establecer un umbral voxel intensidad mínima para reducir la interferencia de la fluorescencia de fondo en el canal de destino. Hemos definido este umbral empíricamente en 9000 au Usando una lista de parámetros disponibles en la aplicación de medición, hemos cuantificado el volumen de epitelio en continuidad con el núcleo original (demarcada en el canal de autofluorescentes). Esto nos permitió cuantificar los volúmenes de derivación epitelial entre diferentes las muestras tumorales. </li></ol> 6. Los resultados representativos: Durante el desarrollo de la prueba, un total de cáncer de mama 52 muestras de biopsia con una amplia gama de histopatología se han utilizado. De estos &lt;10% (5 / 52) no ha facilitado al menos un ensayo de invasión viable. El fracaso del ensayo se debe a bacterias o supra-infección o material insuficiente la biopsia del tumor. Por lo tanto la aplicación de la prueba no se limita a un subtipo específico de cáncer de mama. Ensayos típicos (figura 1) fija después de 20 días en la cultura de colágeno y posteriormente teñidos para citoqueratina. Volocity software es capaz de reconocer tanto la mayor parte del epitelio continuo con el núcleo original y los grupos separados de células invasoras y cuantificar cada volumen por separado. <img src="/files/ftp_upload/3085/3085fig1.jpg" alt="Figura 1" /> Figura 1. Invasión ensayos posteriores a la fijación y cuantificación. El ensayo apoya las muestras obtenidas de múltiples subtipos de cáncer de mama: a) ER + b) HER2 + y c) ER-

<ol>
	<li>Lopez-Garcia, M. A., Geyer, F. C., Lacroix-Triki, M., Marchio, C., Reis-Filho, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+cancer+precursors+revisited:+molecular+features+and+progression+pathways.+Histopathology.">Breast cancer precursors revisited: molecular features and progression pathways. Histopathology.</a> 57, 171-192 (2010).</li><li>Sabeh, F., Shimizu-Hirota, R., Weiss, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protease-dependent+versus+-independent+cancer+cell+invasion+programs:+three-dimensional+amoeboid+movement+revisited.">Protease-dependent versus -independent cancer cell invasion programs: three-dimensional amoeboid movement revisited.</a> J Cell Biol. 185, 11-19 (2009).</li><li>Dabrosin, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Increased+extracellular+local+levels+of+estradiol+in+normal+breast+in+vivo+during+the+luteal+phase+of+the+menstrual+cycle.">Increased extracellular local levels of estradiol in normal breast in vivo during the luteal phase of the menstrual cycle.</a> J Endocrinol. 187, 103-108 (2005).</li><li>Alanentalo, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tomographic+molecular+imaging+and+3D+quantification+within+adult+mouse+organs.">Tomographic molecular imaging and 3D quantification within adult mouse organs.</a> Nat Methods. 4, 31-33 (2007).</li><li>Sharpe, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optical+projection+tomography+as+a+tool+for+3D+microscopy+and+gene+expression+studies.">Optical projection tomography as a tool for 3D microscopy and gene expression studies.</a> Science. 296, 541-545 (2002).</li><li>Hidalgo-Carcedo, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+requires+suppression+of+actomyosin+at+cell-cell+contacts+mediated+by+DDR1+and+the+cell+polarity+regulators+Par3+and+Par6.">Collective cell migration requires suppression of actomyosin at cell-cell contacts mediated by DDR1 and the cell polarity regulators Par3 and Par6.</a> Nat Cell Biol. 13, 49-58 (2011).</li><li>Katz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gene+on+the+HER2+amplicon,+C35,+is+an+oncogene+in+breast+cancer+whose+actions+are+prevented+by+inhibition+of+Syk.">A gene on the HER2 amplicon, C35, is an oncogene in breast cancer whose actions are prevented by inhibition of Syk.</a> Br J Cancer. 103, 401-410 (2010).</li><li>Timpson, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+Regulation+of+RhoA+Activity+during+Pancreatic+Cancer+Cell+Invasion+Driven+by+Mutant+p53.">Spatial Regulation of RhoA Activity during Pancreatic Cancer Cell Invasion Driven by Mutant p53.</a> Cancer Res. 71, 747-757 (2011).</li></ol>

SEW y JF son empleados por MRC Technology, que está comercializando Bioptonics escáneres OPT. Otros autores declaran no tener ningún conflicto de intereses.

A base de colágeno ensayos para estudiar el comportamiento de líneas celulares de cáncer son ampliamente utilizados 6,7,8. Sin embargo, estos ensayos no han reflejado en su totalidad la complejidad del tumor y su entorno. En este estudio, mostramos que los materiales de cáncer de mama humano puede ser utilizado de una manera similar, ex vivo. Además, OPT es una herramienta útil para caracterizar la expansión 3D de cáncer de mama. Se encontró que la principal limitación de esta técnica es la disponibilidad de materiales tumor (una sola biopsia con aguja gruesa es suficiente para 4-6 ensayos). Otro factor limitante es que la invasión estructural sólo se produce en aproximadamente el 40% de los ensayos. Para superar esto, que regularmente agregar cualquier tratamiento deseado alrededor del día 7 a todos los ensayos con un crecimiento visible. El uso de los territorios palestinos ocupados podría ampliarse en el futuro para detectar también cambios masivos, ex vivo, que resulta del tratamiento de drogas. Esto a su vez podría facilitar el tratamiento del cáncer más individualizada, independiente de los modelos animales. Actualmente estamos explorando la respuesta de los tumores RE + al tamoxifeno ex vivo.

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Catálogo N º </td></tr><tr><td> Completa MEGM </td><td> Lonza </td><td> CC-3150 </td></tr><tr><td> DMEM/F12 </td><td> Sigma-Aldrich </td><td> D8900 10X1L </td></tr><tr><td> β-estradiol </td><td> Sigma-Aldrich </td><td> E8875-250MG </td></tr><tr><td> Conejo anti-citoqueratina </td><td> Dako </td><td> Z0622 </td></tr><tr><td> Cabra anti-conejo Alexa555 </td><td> Invitrogen </td><td> A21422 </td></tr></tbody></table> Todos los reactivos otra norma se obtuvieron de Sigma-Aldrich

long-term culture of human breast cancer specimens and their analysis using optical projection tomography

El cáncer de mama es la principal causa de mortalidad en el mundo occidental. Es bien sabido que la propagación del cáncer de mama, en primer lugar a nivel local y distal más tarde, es un factor importante en el pronóstico del paciente. Sistemas experimentales de cáncer de mama se basan en las líneas celulares derivadas de tumores por lo general primario o derrames pleurales. Dos grandes obstáculos dificultan esta investigación: (i) algunos conocidos subtipos de cáncer de mama (tumores pronóstico notablemente pobre luminal B) no están representadas en colecciones de la línea actual, (ii) la influencia del microambiente tumoral no se suele tener en cuenta. Se demuestra una técnica de cultivo primario especímenes de cáncer de mama de todos los subtipos. Esto se logra mediante el uso de tres dimensiones (3D) de los sistemas de cultivo en el que pequeños trozos de tumor están incrustados en el colágeno de rata suave que amortigua. El plazo de 2-3 semanas, las células tumorales se propagan en el colágeno y la forma de diversas estructuras similares a los observados en tumours1 humanos. Adipocitos viables, las células epiteliales y fibroblastos en el núcleo original se hicieron evidentes en la histología. Células epiteliales malignos con morfología escamoide se demostró en la invasión de colágeno circundante. Pleomorfismo nuclear fue evidente dentro de estas células, junto con figuras de mitosis y cuerpos apoptóticos. Hemos empleado la tomografía óptica de proyección (OPT), una tecnología de imagen 3D, con el fin de cuantificar el grado de diseminación del tumor en la cultura. Hemos utilizado OPT para medir el volumen de la masa de la cultura del tumor, un parámetro de una medida rutinaria en el tratamiento neo-adyuvante de pacientes con cáncer de mama para evaluar la respuesta al tratamiento farmacológico. Aquí, se presenta una oportunidad para los tumores de mama humanos sin cultura sub-tipo de prejuicios y cuantificar la propagación de los ex vivo. Este método podría ser utilizado en el futuro para cuantificar la sensibilidad de la droga en el tumor original. Esto puede proporcionar un modelo más predictivo que las líneas celulares actualmente en uso.

Watch this Scientific Journal Video about Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography at JoVE.com

Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Hemos desarrollado una base de colágeno en el ensayo in vitro, que promueve la proliferación y la invasión de las muestras de todos los subtipos de cáncer de mama. Tomografía óptica de proyección, una técnica de tres dimensiones se utilizó la microscopía para visualizar y cuantificar la expansión del tumor. Este ensayo puede ser utilizado para cuantificar la respuesta al fármaco de las muestras tumorales individuales.

A largo plazo la cultura de muestras humanas del cáncer de pecho y su análisis mediante tomografía óptica de proyección

Breast cancer is a leading cause of mortality in the Western world. It is well established that the spread of breast cancer, first locally and later ...

long-term-culture-human-breast-cancer-specimens-their-analysis-using

Research

JoVE Journal

Biology

14.6K vistas.  University of Edinburgh. Hemos desarrollado una base de colágeno en el ensayo in vitro, que promueve la proliferación y la invasión de las muestras de todos los subtipos de cáncer de mama. Tomografía óptica de proyección, una técnica de tres dimensiones se utilizó la microscopía para visualizar y cuantificar la expansión del tumor. Este ensayo puede ser utilizado para cuantificar la respuesta al fármaco de las muestras tumorales individuales.

Video: Long-term Culture of Human Breast Cancer Specimens and Their Analysis Using Optical Projection Tomography

Long-term Imaging Mammalian Cells using Wide-Field Microscopy

A largo plazo de imágenes usando las células de mamíferos Wide-Field Microscopía

Investigación

Biología

A Neuronal and Astrocyte Co-Culture Assay for High Content Analysis of Neurotoxicity

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing measurement of multiple cellular phenotypes within a single assay. In this study, we utilized HCA to develop a novel assay for neurotoxicity. Neurotoxicity assessment represents an important part of drug safety evaluation, as well as being a significant focus of environmental protection efforts. Additionally, neurotoxicity is also a well-accepted in vitro marker of the development of neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. Recently, the application of HCA to neuronal screening has been reported. By labeling neuronal cells with &beta;III-tubulin, HCA assays can provide high-throughput, non-subjective, quantitative measurements of parameters such as neuronal number, neurite count and neurite length, all of which can indicate neurotoxic effects. However, the role of astrocytes remains unexplored in these models. Astrocytes have an integral role in the maintenance of central nervous system (CNS) homeostasis, and are associated with both neuroprotection and neurodegradation when they are activated in response to toxic substances or disease states. GFAP is an intermediate filament protein expressed predominantly in the astrocytes of the CNS. Astrocytic activation (gliosis) leads to the upregulation of GFAP, commonly accompanied by astrocyte proliferation and hypertrophy. This process of reactive gliosis has been proposed as an early marker of damage to the nervous system. The traditional method for GFAP quantitation is by immunoassay. This approach is limited by an inability to provide information on cellular localization, morphology and cell number. We determined that HCA could be used to overcome these limitations and to simultaneously measure multiple features associated with gliosis - changes in GFAP expression, astrocyte hypertrophy, and astrocyte proliferation - within a single assay. In co-culture studies, astrocytes have been shown to protect neurons against several types of toxic insult and to critically influence neuronal survival. Recent studies have suggested that the use of astrocytes in an in vitro neurotoxicity test system may prove more relevant to human CNS structure and function than neuronal cells alone. Accordingly, we have developed an HCA assay for co-culture of neurons and astrocytes, comprised of protocols and validated, target-specific detection reagents for profiling &beta;III-tubulin and glial fibrillary acidic protein (GFAP). This assay enables simultaneous analysis of neurotoxicity, neurite outgrowth, gliosis, neuronal and astrocytic morphology and neuronal and astrocytic development in a wide variety of cellular models, representing a novel, non-subjective, high-throughput assay for neurotoxicity assessment. The assay holds great potential for enhanced detection of neurotoxicity and improved productivity in neuroscience research and drug discovery.

This article describes a novel protocol and reagent set designed for sensitive measurement of neurotoxic effects of compounds and treatments on co-cultures of neurons and astrocytes using high content analysis. Results demonstrate that high content analysis represents an exciting novel technology for neurotoxicity assessment.

A neuronales y astrocitos Co-cultivo de ensayo para el análisis de alto contenido de neurotoxicidad

High Content Analysis (HCA) assays combine cells and detection reagents with automated imaging and powerful image analysis algorithms, allowing ...

Born Normalization for Fluorescence Optical Projection Tomography for Whole Heart Imaging

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental biology and gene expression studies. The technique renders biological sample optically transparent by first dehydrating them and then placing in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution). The technique renders biological samples optically transparent by first dehydrating them in graded ethanol solutions then placing them in a mixture of benzyl alcohol and benzyl benzoate in a 2:1 ratio (BABB or Murray s Clear solution) to clear. After the clearing process the scattering contribution in the sample can be greatly reduced and made almost negligible while the absorption contribution cannot be eliminated completely. When trying to reconstruct the fluorescence distribution within the sample under investigation, this contribution affects the reconstructions and leads, inevitably, to image artifacts and quantification errors.. While absorption could be reduced further with a permanence of weeks or months in the clearing media, this will lead to progressive loss of fluorescence and to an unrealistically long sample processing time. This is true when reconstructing both exogenous contrast agents (molecular contrast agents) as well as endogenous contrast (e.g. reconstructions of genetically expressed fluorescent proteins).

We suggest a Born normalized approach for Optical Projection Tomography (BnOPT) that accounts for the absorption properties of imaged samples to obtain accurate and quantitative fluorescence tomographic reconstructions. We use the proposed algorithm to reconstruct the fluorescence molecular probe distribution within small animal organs.

La normalización nació para tomografía de fluorescencia de proyección óptica de imágenes de todo corazón

Optical projection tomography is a three-dimensional imaging technique that has been recently introduced as an imaging tool primarily in developmental ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Aislamiento y cultivo de células madre adultas epiteliales de la piel humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo Cultura de células humanas primarias epiteliales del tubo de Falopio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish water and sediment. The choice of biological indicators must be based on reliable scientific knowledge and, possibly, on the availability of standardized procedures. In this article, we present a standardized protocol that used the marine crustacean Artemia to evaluate the toxicity of chemicals and/or of marine environmental matrices. Scientists propose that the brine shrimp (Artemia) is a suitable candidate for the development of a standard bioassay for worldwide utilization. A number of papers have been published on the toxic effects of various chemicals and toxicants on brine shrimp (Artemia). The major advantage of this crustacean for toxicity studies is the overall availability of the dry cysts; these can be immediately used in testing and difficult cultivation is not demanded1,2. Cyst-based toxicity assays are cheap, continuously available, simple and reliable and are thus an important answer to routine needs of toxicity screening, for industrial monitoring requirements or for regulatory purposes3. The proposed method involves the mortality as an endpoint. The numbers of survivors were counted and percentage of deaths were calculated. Larvae were considered dead if they did not exhibit any internal or external movement during several seconds of observation4. This procedure was standardized testing a reference substance (Sodium Dodecyl Sulfate); some results are reported in this work. This article accompanies a video that describes the performance of procedural toxicity testing, showing all the steps related to the protocol.

Long-term Lethal Toxicity Test with the Crustacean Artemia franciscana

This study concerns the development and standardization of a valuable methodological protocol to determine long-term (14 days) lethal toxicity exerted by chemical substances, industrial wastewater or sewage and liquid environmental samples on the saltwater crustacean, Artemia franciscana.

A largo plazo Prueba de toxicidad letal con el Crustáceo Artemia franciscana</em

Our research activities target the use of biological methods for the evaluation of environmental quality, with particular reference to saltwater/brackish ...

Serial Enrichment of Spermatogonial Stem and Progenitor Cells (SSCs) in Culture for Derivation of Long-term Adult Mouse SSC Lines

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly the lifetime of the animal. While the precise mechanisms that govern self-renewal and differentiation in vivo are challenging to study, various systems have been developed previously to propagate murine SSCs in vitro using a combination of specialized culture media and feeder cells1-3.
Most in vitro forays into the biology of SSCs have derived cell lines from neonates, possibly due to the difficulty in obtaining adult cell lines4. However, the testis continues to mature up until ~5 weeks of age in most mouse strains. In the early post-natal period, dramatic changes occur in the architecture of the testis and in the biology of both somatic and spermatogenic cells, including alterations in expression levels of numerous stem cell-related genes. Therefore, neonatally-derived SSC lines may not fully recapitulate the biology of adult SSCs that persist after the adult testis has reached a steady state.
Several factors have hindered the production of adult SSC lines historically. First, the proportion of functional stem cells may decrease during adulthood, either due to intrinsic or extrinsic factors5,6. Furthermore, as with other adult stem cells, it has been difficult to enrich SSCs sufficiently from total adult testicular cells without using a combination of immunoselection or other sorting strategies7. Commonly employed strategies include the use of cryptorchid mice as a source of donor cells due to a higher ratio of stem cells to other cell types8. Based on the hypothesis that removal of somatic cells from the initial culture disrupts interactions with the stem cell niche that are essential for SSC survival, we previously developed methods to derive adult lines that do not require immunoselection or cryptorchid donors but rather employ serial enrichment of SSCs in culture, referred to hereafter as SESC2,3.
The method described below entails a simple procedure for deriving adult SSC lines by dissociating adult donor seminiferous tubules, followed by plating of cells on feeders comprised of a testicular stromal cell line (JK1)3. Through serial passaging, strongly adherent, contaminating non-germ cells are depleted from the culture with concomitant enrichment of SSCs. Cultures produced in this manner contain a mixture of spermatogonia at different stages of differentiation, which contain SSCs, based on long-term self renewal capability. The crux of the SESC method is that it enables SSCs to make the difficult transition from self-renewal in vivo to long-term self-renewal in vitro in a radically different microenvironment, produces long-term SSC lines, free of contaminating somatic cells, and thereby enables subsequent experimental manipulation of SSCs.

Serial Enrichment of Spermatogonial Stem and Progenitor Cells SSCs in Culture for Derivation of Long-term Adult Mouse SSC Lines

A simple method to derive and maintain spermatogonial stem and progenitor cell lines from adult mice is presented here. The method utilizes feeder cells originating from the somatic cell compartment of the adult mouse testis. This technique is applicable to common mouse strains, including transgenic, knock-out, and knock-in mice.

Enriquecimiento de serie de la madre de espermatogonias y células progenitoras (SSC) en Cultura para la derivación de largo plazo de ratones adultos Líneas SSC

Spermatogonial stem and progenitor cells (SSCs) of the testis represent a classic example of adult mammalian stem cells and preserve fertility for nearly ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

La detección de alteraciones genéticas somáticas en muestras de tumores por Exon de captura y secuenciación masivamente paralelo

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

The development of zebrafish can be followed over days with light sheet microscopy when embryos are embedded in optically clear polymer tubes with low-concentration agarose.

Multicapa de montaje para microscopía de luz Hoja de largo plazo del pez cebra

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is ...

Isolation, Transfection, and Long-Term Culture of Adult Mouse and Rat Cardiomyocytes

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. Adult mammalian CMs are terminally differentiated cells with minimal proliferative capacity. The post-mitotic state of adult CMs not only restricts cardiomyocyte cell cycle progression but also limits the efficient culture of CMs. Moreover, the long-term culture of adult CMs is necessary for many studies, such as CM proliferation and analysis of gene expression.
The mouse and the rat are the two most preferred laboratory animals to be used for cardiomyocyte isolation. While the long-term culture of rat CMs is possible, adult mouse CMs are susceptible to death and cannot be cultured more than five days under normal conditions. Therefore, there is a critical need to optimize the cell isolation and long-term culture protocol for adult murine CMs. With this modified protocol, it is possible to successfully isolate and culture both adult mouse and rat CMs for more than 20 days. Moreover, the siRNA transfection efficiency of isolated CM is significantly increased compared to previous reports. For adult mouse CM isolation, the Langendorff perfusion method is utilized with an optimal enzyme solution and sufficient time for complete extracellular matrix dissociation. In order to obtain pure ventricular CMs, both atria were dissected and discarded before proceeding with the disassociation and plating. Cells were dispersed on a laminin coated plate, which allowed for efficient and rapid attachment. CMs were allowed to settle for 4-6 h before siRNA transfection. Culture media was refreshed every 24 h for 20 days, and subsequently, CMs were fixed and stained for cardiac-specific markers such as Troponin and markers of cell cycle such as KI67.

Here, we present a protocol for the isolation, transfection, and long-term culture of adult mouse and rat cardiomyocytes.

Aislamiento, Transfección y Cultura a Largo Plazo de Cardiomiocitos adultos de ratón y rata

Ex vivo culture of the adult mammalian cardiomyocytes (CMs) presents the most relevant experimental system for the in vitro study of cardiac biology. ...