Este estudio fue apoyado por becas a YMS de los Institutos Nacionales de Salud (CA148828), la Universidad de Michigan Gastrointestinal Peptide Center, y Jeffrey A. Colby Colon Cancer Research y los Fondos Liu Tom Memorial del Centro Integral del Cáncer de la Universidad de Michigan.

1. Aislamiento del tumor de colon y de disociación celular <ol><li> Tumores intestinales se pueden aislar a partir de cualquier modelo de cáncer de colon esporádico o inducida por el tratamiento. Los ratones deben ser sometidos a eutanasia con CO 2. A continuación se recogen dos puntos, enrojecida por el frío salina tamponada con fosfato (PBS) y se abren longitudinalmente. Identificar las regiones que contienen los tumores utilizando un microscopio estereoscópico, diseccionar con un par de tijeras, y lavar con PBS frío. </li><li> Incubar los fragmentos que contienen los tumores intestinales en tampón de quelación con EDTA (2 mM de EDTA, 5,6 mmol / L de Na 2 HPO 4, 8,0 mmol / L de KH 2 PO 4, 96.2 mmol / l de NaCl, 1,6 mmoles / L de KCl, 43,4 mmol / L de sacarosa, 54,9 mmol / L de D-sorbitol, 0,5 mmoles / L de DL-ditiotreitol en agua destilada) durante 60 min en hielo. </li><li> Después de quelación, la mayor parte de las células epiteliales intestinales normales será separado, mientras que las células tumorales se permanecer unidos a la mesénquima. Aspirar el tampón de quelación que contiene las células epiteliales normalesy lavar el tumor remanente fragmenta una vez más con Buffer 5 quelación frío ml. </li><li> Aspirar el tampón de quelación, lavar fragmentos tumorales con 5 ml 1x PBS frío. </li><li> Aspirar el PBS 1x, se incuban fragmentos de tumor en tampón de digestión (2,5% de suero bovino fetal, 1 unidad / ml de penicilina, 1 mg / ml de estreptomicina, y 2,5 ng / ml de anfotericina B, 200 U / ml de colagenasa de tipo IV, 125 g / ml de tipo II en dispasa de Eagle modificado por Dulbecco) durante 2 horas a 37 ° C. </li><li> Deje que el fragmento de tumor se asiente por gravedad normal, durante 1 minuto, y recoger el sobrenadante en un tubo de 15 ml halcón. Se precipitan los sola célula tumoral suspensión sobrenadante por centrifugación a 200 xg durante 3 min y lavar una vez con 5 ml de PBS por centrifugación a 200 xg durante 3 min. </li></ol> 2. Cultura de tumor intestinal <ol><li> Resuspender el sedimento de células del tumor con 500 l de PBS, contar las células tumorales individuales aislados usando un hemocitómetro. </li><li> Pellet células tumorales por cientorifuging a 200 xg durante 3 minutos, y volver a suspender en 5 mg / ml de Matrigel en hielo y la placa en placas de 24 pocillos a 15.000 células por 50 l de Matrigel por pocillo. </li><li> Deje que el Matrigel polimerizar durante 15 min a 37 ° C, y añadir 500 l / pocillo basal medio de cultivo (1 unidad / ml de penicilina, 1 mg / ml de estreptomicina, y 2,5 ng / ml de anfotericina B, 10 mmol / L de HEPES , Glutamax 2 mM, 1x suplemento N2, 1x suplemento B27, 1 mmol / L de N-acetilcisteína en Avanzada de Eagle modificado por Dulbecco medium/F12) que contiene 50 ng / ml de EGF murino. </li></ol> 3. Mantenimiento de Organoides establecidos <ol><li> Cambio de medio de cultivo basal conteniendo EGF cada 2 días y paso organoides 1:05 una vez a la semana. </li><li> Para pases, sustituir el medio de cultivo con medio de cultivo basal fresco. Mecánicamente interrumpir organoids y Matrigel utilizando una pipeta P1000 con puntas cortadas y transferir a un tubo de 15 ml halcón. Además disociación mecánica se realiza mediante un tubo de Pasteur pulida al fuegotte. </li><li> Lavar organoides disociadas con 5 ml de medio de cultivo basal y se centrifuga a 200 xg durante 2 min. </li><li> Descartar el sobrenadante, resuspender el precipitado con Matrigel y añadir medio de cultivo como se describe anteriormente. </li></ol> 4. Almacenamiento y recuperación de Organoides establecidos <ol><li> Para el almacenamiento a largo plazo, congelar organoides en N2 líquido que son estables durante al menos 2 años. Para organoids congelación, interrumpir el uso de una pipeta P1000 con puntas cortadas y transferir a un tubo de 15 ml halcón. </li><li> Lavar organoides disociadas con 5 ml de medio de cultivo basal y se centrifuga a 200 xg durante 2 min. </li><li> Descartar el sobrenadante, resuspender el precipitado con de Eagle modificado por Dulbecco (DMEM) que contenía 20% de suero bovino fetal (FBS) y 10% de dimetilsulfóxido (DMSO). </li><li> Células de transferencia en criotubos de 1,5 ml, a continuación, poner los tubos en un recipiente Mr. Frosty congelación Nalgene y guardar en un congelador de -80 º C para alcanzar una velocidad de enfriamientode -1 º C / min. Después de la incubación durante la noche, traslado tubos en N2 líquido. </li><li> Para la recuperación, los organoides congelados se descongelan rápidamente en un 37 ° C baño de agua, lavar con medio de cultivo basal, la desaceleración, resuspender en Matrigel y la cultura de las condiciones descritas anteriormente. </li></ol> 5. Extracción de ARN, extracción de proteínas y Inmunohistoquímica <ol><li> Extracción de ARN: Las células se recogen como se ha descrito anteriormente para el paso. Se aísla el ARN con PicoPure TM ARN kit de aislamiento de acuerdo con las instrucciones del fabricante. </li><li> Extracción de proteínas: Los sedimentos celulares se recolectan como se describió anteriormente y se lisaron en 100 l de tampón de ensayo de radioinmunoprecipitación (RIPA; 50 mmol / l de Tris-HCl pH 7,5, 150 mmol / l de NaCl, 2 mmol / l de EDTA, 1% NP-40, 0.1 % SDS) con 1 inhibidor de la proteasa. </li><li> Inmunohistoquímica: Aspirar el medio de cultivo celular, transferir organoids tumorales con P1000 pipeta con puntas cortadas en Cryo-molde y congelar inmediatamente en tisdemandar medio de congelación (OCT) con hielo seco. Las secciones congeladas se cortaron a 7 micras y se tiñeron como se ha descrito previamente 26. </li></ol>

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No tenemos nada que revelar.

Los procedimientos experimentales descritos en este protocolo permitirán para aislamiento y cultivo de los tumores de colon primarios murinos. El protocolo es una adaptación del trabajo seminal realizado por el Dr. grupo Clevers 1,24,27. Hemos optimizado el tiempo de digestión y la concentración de colagenasa para obtener un mejor rendimiento de organoides tumorales. Los pasos críticos incluyen la digestión tumor de células en células individuales, Matrigel resuspensión, y cultivo selectivo. Para la ...

Las células epiteliales intestinales proliferan y se entreguen a un ritmo extraordinario, superando a todos los otros tejidos en el cuerpo vertebrado 2,3. Las células que se dividen como las células madre intestinales (ISC) y células de tránsito de amplificación se diferencian en cualquiera de las células o los enterocitos 3 secretora (copa, Paneth y enteroendocrinas). El ISC se encuentra en la base de la cripta. Células de Paneth se mueven hacia abajo a la parte inferior de las criptas y son de larga vida, mientras que otros linajes migran hacia arriba a las vellosidades 3,4. Aquí las células se exponen a los contenidos del intestino, incluyendo la microbiota y se desprenden de las puntas de las vellosidades a través de un mecanismo de apoptosis inducida por la anoikis. Aunque el colon carece de vellosidades y células de Paneth, el mecanismo para el mantenimiento de la homeostasis es similar 4. La vía de señalización de Wnt se ha implicado en jugar un papel crucial en la proliferación intestinal y mantenimiento ISC 4. Supresión de the TCF4 factor de transcripción, un efector corriente abajo de la señalización de Wnt, conduce a la pérdida de las células madre intestinales y posterior descomposición del tejido 5. Del mismo modo, la expresión transgénica de la DKK1 inhibidor de Wnt reduce la proliferación epitelial y agota linajes de células secretoras 6. Por el contrario, la sobreexpresión de Wnt el agonista R-espondina-1 induce la proliferación rápida y potente de células de las criptas intestinales 7. Dada la importancia de la señalización de Wnt para la homeostasis intestinal, las mutaciones vía Wnt se observan frecuentemente en cáncer de colon 8. El cáncer de colon es la tercera causa principal de muerte por cáncer en Estados Unidos 9. El exceso de ingesta de la dieta con la carne roja y el alcohol, la reducción de la actividad física, y hereda y mutaciones somáticas se considera que son factores de riesgo de cáncer de colon 10,11. La coli (APC) gen poliposis adenomatosa, un factor clave de la señalización de Wnt, se encuentra mutado en la mayoría de los palos pacientes con familiar, esporádica, y el cáncer de colon asociado con la colitis 12,13. Las mutaciones de otros factores implicados en la vía de señalización Wnt incluyendo AXIN2 y β-catenina se observan también en cáncer de colon 14,15. Sin embargo, el mecanismo exacto y un tratamiento eficaz para el cáncer de colon todavía no existen. Para facilitar la investigación de los mecanismos moleculares para el cáncer de colon, colon humano líneas celulares de cáncer que representan diferentes etapas de la progresión del cáncer de un benigna a un tipo de célula agresiva se han establecido 16-18. Ratón de carcinoma de colon líneas celulares con diferentes propiedades metastásicas también están disponibles 19,20. Sin embargo, se prefieren las células primarias o cultivos de organoides a través de líneas celulares transformadas, ya que imitan el estado in vivo y generan más fisiológicamente relevante de datos 21. La mayoría de las líneas celulares derivadas de cáncer de colon crecen como monocapa unida a la placa o como suspensiones de células, lacking de la orientación apical-basolateral y uniones estrechas entre las células. Además, las células epiteliales intestinales normales y tumorales in vivo se someten a una forma espontánea de la apoptosis anoikis denominado como las células diferenciadas alcanzan las puntas de las vellosidades y se desprenden 22. Estas características son difíciles de recapitular en líneas celulares, pero son importantes en el proceso de desarrollo de cáncer de colon 23. Estas características se mantienen en organoides primarios. Además, los cultivos de organoides tumorales proporcionan un sistema eficaz para evaluar las funciones autónomas tumorales de genes en comparación con los estudios in vivo. La manipulación genética in vivo del intestino es un proceso que consume tiempo principalmente a través de la creación de transgénicos y / o ratones knockout el uso de controladores delgado-específicos. Sin embargo, los organoides tumorales son fácilmente susceptible de manipulaciones genéticas mediadas virales y por lo tanto una gran herramienta para la evaluación de los mecanismos moleculares precisos. Intestinales culturas organoides tumorales primarias VHAe ha demostrado ser una técnica segura y eficaz. Cultivo celular intestinal primaria puede establecer organoids intestinales funcionales, con estructura de las criptas-vellosidades in vitro a partir de un solo adulto LGR5 + células madre 24. Estos organoides pueden ser trasplantados y implantadas en tejido de colon dañado para la regeneración 25. Además la adaptación de las condiciones de cultivo había hecho organoides epiteliales similares de colon de ratón y el intestino delgado y el colon factible 1 humana. Para el cultivo primario de colon normal epitelio, medio de cultivo basal, así como factores de crecimiento, incluyendo EGF, Noggin, R-espondina y Wnt3a son esenciales, mientras que el medio de cultivo basal y EGF es suficiente para el cultivo de ratón de colon organoides tumor primario 1. Aquí se describe un protocolo detallado para aislar, la cultura, y generar organoids tumor de colon.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Matrigel Basement Membrane Matrix</td>
			<td>BD Biosciences</td>
			<td>356234</td>
			<td>5 mg/ml</td>
		</tr>
		<tr>
			<td>Collagenase Type IV</td>
			<td>Worthington</td>
			<td>LS004188</td>
			<td>375 U/mg</td>
		</tr>
		<tr>
			<td>Dispase</td>
			<td>Gibco</td>
			<td>17105-041</td>
			<td>1.8 U/mg</td>
		</tr>
		<tr>
			<td>Advanced DMEM/F12</td>
			<td>Invitrogen</td>
			<td>12634010</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Epidermal Growth Factor (EGF), Murine, Natural</td>
			<td>Invitrogen</td>
			<td>53003-018</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>N2 Supplement</td>
			<td>Invitrogen</td>
			<td>17502-048</td>
			<td>100 x</td>
		</tr>
		<tr>
			<td>B27 Supplement</td>
			<td>Invitrogen</td>
			<td>17504-044</td>
			<td>50 x</td>
		</tr>
		<tr>
			<td>Glutamax-I</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td>100x</td>
		</tr>
		<tr>
			<td>N-Acetylcysteine</td>
			<td>Sigma</td>
			<td>A9165-5G</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Dulbecco's Modified Eagle Medium</td>
			<td>Invitrogen</td>
			<td>11965-092</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>PicoPureTM RNA Isolation Kit</td>
			<td>Invitrogen</td>
			<td>KIT0204</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

in vitro organoid culture of primary mouse colon tumors

Varias líneas celulares de cáncer de colon humano y murino se han establecido, la integridad fisiológica de los tumores de colon tales como múltiples capas de células, de polaridad basal-apical, la capacidad para diferenciar, y anoikis no se mantienen en el cáncer de colon líneas celulares derivadas. El presente estudio demuestra un método para el cultivo de ratón primarios de colon organoides tumorales adaptadas de Sato, T. et. Al 1, que conserva importantes funciones fisiológicas de los tumores de colon. Este método consiste en la recopilación ratón de tumor de colon tejido, adyacente normal del colon epitelio disociación, las células de tumor de colon digestión en células individuales, la incorporación de células de tumor de colon en matrigel, y cultivo selectivo basado en el principio de que las células tumorales mantener el crecimiento en condiciones de limitación de nutrientes en comparación a la normalidad células epiteliales. Los organoids tumor primario si aisladas de ratones modificados genéticamente ofrecen un sistema muy útil para evaluar func autónoma tumoriones de genes específicos. Por otra parte, los organoides tumorales son susceptibles a la manipulación genética de virus meditado entrega de genes, por lo tanto, las vías de señalización implicadas en la tumorigénesis de colon podrían ser investigados exhaustivamente por la sobreexpresión o derribo. Organoids Primary cultura tumor proporciona un medio fisiológico pertinentes y viables para el estudio de los mecanismos y modalidades terapéuticas para la tumorigénesis de colon.

El curso temporal de la formación de un tumor de colon organoide a partir de una de tres meses de edad, APC Min / + ratón se muestra en la Figura 1. En el día 0, las células individuales se pudieron observar varias horas después de chapado (Figura 1A). En el día 1, sobrevivieron de colon células epiteliales tumorales con núcleos refractario se pudo observar. En el día 3, el tamaño de las células se duplicó. En el día 6, el tamaño de organoide...

Watch this Scientific Journal Video about In vitro Organoid Culture of Primary Mouse Colon Tumors at JoVE.com

In vitro Organoid Culture of Primary Mouse Colon Tumors

Un método simple para establecer organoide del tumor de colon murino primaria se describe. Este método utiliza la característica de que las células de tumor de colon sobrevivir y crecer en organoides en medios que contienen factores de crecimiento limitadas, mientras que el colon normal epiteliales no lo hacen.

In vitro organoide Cultura de tumores de colon primarios Ratón

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

in-vitro-organoid-culture-of-primary-mouse-colon-tumors

Research

JoVE Journal

Medicine

In vitro Organoid Culture of Primary Mouse Colon Tumors

34.9K vistas.  University of Michigan. Un método simple para establecer organoide del tumor de colon murino primaria se describe. Este método utiliza la característica de que las células de tumor de colon sobrevivir y crecer en organoides en medios que contienen factores de crecimiento limitadas, mientras que el colon normal epiteliales no lo hacen.

Video: In vitro Organoid Culture of Primary Mouse Colon Tumors

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited time. These cultured tumorspheres may exhibit markedly different cellular phenotypes when compared to the original tumors. Demonstration that cultured cells have the capability of forming new tumors is important to ensure that cultured cells model the biology of the original tumor. 
Here we present a protocol for propagating human retinoblastoma tumors in vivo using Rag2-/- immune deficient mice. Cultured human retinoblastoma tumorspheres of low passage or cells obtained from freshly harvested human retinoblastoma tumors injected directly into the vitreous cavity of murine eyes form tumors within 2-4 weeks. These tumors can be harvested and either further passaged into murine eyes in vivo or grown as tumorspheres in vitro. Propagation has been successfully carried out for at least three passages thus establishing a continuing source of human retinoblastoma tissue for further experimentation. 
Wesley S. Bond and Lalita Wadhwa are co-first authors.

A method is described to propagate human retinoblastoma tumors in mice. Tumor cells are directly injected into the eyes of immune deficient mice. Secondary tumors have been successfully established using both cells directly harvested from human tumors and cultured tumorspheres.

Establecimiento y propagación de los tumores del retinoblastoma humanos en ratones con deficiencia inmune

Culturing retinoblastoma tumor cells in defined stem cell media gives rise to primary tumorspheres that can be grown and maintained for only a limited ...

Investigación

Medicina

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Piel biopsia en sacabocados explante cultura para Derivación de fibroblastos humanos primarios

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in either sporadic or familial (neurofibromatosis type 2, NF2) forms, both associated with inactivating defects in the NF2 tumor suppressor gene. Treatment for VSs is generally surgical resection or radiosurgery, however the morbidity of such procedures has driven investigations into less invasive treatments. Historically, lack of access to fresh tissue specimens and the fact that schwannoma cells are not immortalized have significantly hampered the use of primary cultures for investigation of schwannoma tumorigenesis. To overcome the limited supply of primary cultures, the immortalized HEI193 VS cell line was generated by transduction with HPV E6 and E7 oncogenes. This oncogenic transduction introduced significant molecular and phenotypic alterations to the cells, which limit their use as a model for human schwannoma tumors. We therefore illustrate a simplified, reproducible protocol for culture of primary human VS cells. This easily mastered technique allows for molecular and cellular investigations that more accurately recapitulate the complexity of VS disease.

Vestibular Schwannomas (VSs) are non-malignant tumors of Schwann cell (SC) origin, associated with mutations in the NF2 tumor suppressor gene. We report a reproducible, efficient protocol for primary human VS cell culture that allows for molecular and cellular experimental manipulation and analysis and recapitulates the heterogeneous nature of human disease.

Cultivo primario de Human vestibular schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both direct and rapid analysis of therapeutic sensitivity and resistance. However, recent evidence suggests that therapeutic response is not exclusive to the inherent molecular composition of cancer cells but rather is greatly influenced by the tumor cell microenvironment, a feature that cannot be recapitulated by traditional culturing methods. Even implementation of tumor xenografts, though providing a wealth of information on drug delivery/efficacy, cannot capture the tumor cell/microenvironment crosstalk (i.e., soluble factors) that occurs within human tumors and greatly impacts tumor response. To this extent, we have developed an ex vivo (fresh tissue sectioning) technique which allows for the direct assessment of treatment response for preclinical and clinical therapeutics development. This technique maintains tissue integrity and cellular architecture within the tumor cell/microenvironment context throughout treatment response providing a more precise means to assess drug efficacy.

Ex Vivo Treatment Response of Primary Tumors and/or Associated Metastases for Preclinical and Clinical Development of Therapeutics

Established cancer cell lines and xenografts have been the mainstay of cancer research for the past several decades. However, recent evidence suggests that therapeutic response is greatly influenced by the tumor cell microenvironment. Therefore, we have developed an ex vivo analysis of primary tumor specimens for drug development purposes.

Ex Vivo respuesta al tratamiento de los tumores primarios y / o Asociados Metástasis de Preclínica y Desarrollo Clínico de Terapéutica

The molecular analysis of established cancer cell lines has been the mainstay of cancer research for the past several decades. Cell culture provides both ...

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The approach allows unbiased identification and functional characterization of individual colorectal afferents. Extracellular recordings of propagated action potentials (APs) that originate from one or a few afferent (i.e., single-fiber) receptive fields (RFs) in the colorectum are made from teased nerve fiber fascicles. The colorectum is removed with either the pelvic (PN) or lumbar splanchnic (LSN) nerve attached and opened longitudinally. The tissue is placed in a recording chamber, pinned flat and perfused with oxygenated Krebs solution. Focal electrical stimulation is used to locate the colorectal afferent endings, which are further tested by three distinct mechanical stimuli (blunt probing, mucosal stroking and circumferential stretch) to functionally categorize the afferents into five mechanosensitive classes. Endings responding to none of these mechanical stimuli are categorized as mechanically-insensitive afferents (MIAs). Both mechanosensitive and MIAs can be assessed for sensitization (i.e., enhanced response, reduced threshold, and/or acquisition of mechanosensitivity) by localized exposure of RFs to chemicals (e.g., inflammatory soup (IS), capsaicin, adenosine triphosphate (ATP)). We describe the equipment and colorectum&#8211;nerve recording preparation, harvest of colorectum with attached PN or LSN, identification of RFs in the colorectum, single-fiber recording from nerve fascicles, and localized application of chemicals to the RF. In addition, challenges of the preparation and application of standardized mechanical stimulation are also discussed.

In vitro Functional Characterization of Mouse Colorectal Afferent Endings

This video demonstrates a protocol for conducting single-fiber electrophysiological recordings on an in vitro mouse colorectum-nerve preparation.

In vitro Caracterización funcional de ratón colorrectal aferente Endings

This video demonstrates in detail an in vitro single-fiber electrophysiological recording protocol using a mouse colorectum-nerve preparation. The ...

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análisis clínico-patológica de los genes miARN expresión en el cáncer de mama mediante el uso de tejidos miARN<em&gt; In Situ</em&gt; La hibridación

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Aislamiento y cultivo de células endoteliales primarias de las arterias y venas caninas

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant CFTR channel in subjects with cystic fibrosis (CF). Identification of subjects that may benefit from these drugs is challenging because of the extensive heterogeneity of CFTR mutations, as well as other unknown factors that contribute to individual drug efficacy. Here, we describe a simple and relatively rapid assay for measuring individual CFTR function and response to CFTR modulators in vitro. Three dimensional (3D) epithelial organoids are grown from rectal biopsies in standard organoid medium. Once established, the organoids can be bio-banked for future analysis. For the assay, 30-80 organoids are seeded in 96-well plates in basement membrane matrix and are then exposed to drugs. One day later, the organoids are stained with calcein green, and forskolin-induced swelling is monitored by confocal live cell microscopy at 37 &#176;C. Forskolin-induced swelling is fully CFTR-dependent and is sufficiently sensitive and precise to allow for discrimination between the drug responses of individuals with different and even identical CFTR mutations. In vitro swell responses correlate with the clinical response to therapy. This assay provides a cost-effective approach for the identification of drug-responsive individuals, independent of their CFTR mutations. It may also be instrumental in the development of future CFTR modulators.

Forskolin-induced Swelling in Intestinal Organoids: An In Vitro Assay for Assessing Drug Response in Cystic Fibrosis Patients

This protocol describes an assay for measuring CFTR function and CFTR modulator responses in cultured tissue from subjects with cystic fibrosis (CF). Biopsy-derived intestinal organoids swell in a cAMP-driven fashion, a response that is defective (or strongly reduced) in CF organoids and can be restored by exposure to CFTR modulators.

Inducida por la forskolina Hinchazón en organoides intestinal: Una<em&gt; In Vitro</em&gt; Ensayo para evaluar la respuesta de drogas en pacientes con fibrosis quística

Recently-developed cystic fibrosis transmembrane conductance regulator (CFTR)-modulating drugs correct surface expression and/or function of the mutant ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Los desequilibrios musculares: Pruebas y entrenamiento de fuerza funcional excéntrico isquiotibiales en poblaciones deportivas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

On-Site Sampling and Extraction of Brain Tumors for Metabolomics and Lipidomics Analysis

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still in need. In recent years, mass spectrometry has become a method of choice for untargeted profiling of discriminatory compound as potential biomarkers of a disease. Biofluids are generally considered as preferable matrices given their accessibility and easier sample processing while direct tissue profiling provides more selective information about a given cancer. Preparation of tissues for the analysis via traditional methods is much more complex and time-consuming, and, therefore, not suitable for fast on-site analysis. The current work presents a protocol combining sample preparation and extraction of small molecules on-site, immediately after tumor resection. The sampling device, which is of the size of an acupuncture needle, can be inserted directly into the tissue and then transported to the nearby laboratory for instrumental analysis. The results of metabolomics and lipidomics analyses demonstrate the capability of the approach for the establishment of phenotypes of tumors related to the histological origin of the tumor, malignancy, and genetic mutations, as well as for the selection of discriminating compounds or potential biomarkers. The non-destructive nature of the technique permits subsequent performance of routinely used tests e.g., histological tests, on the same samples used for SPME analysis, thus enabling attainment of more comprehensive information to support personalized diagnostics.

The manuscript presents on-site sampling of human brain tumors with solid phase microextraction followed by their biochemical profiling towards the discovery of biomarkers.

Muestreo y extracción in situ de tumores cerebrales para el análisis de metabolómica y lipidómica

Despite the variety of tools available for cancer diagnosis and classification, methods that enable fast and simple characterization of tumors are still ...