La financiación de este proyecto está a cargo de la Fundación Clayton para la Investigación y la Fundación Retina investigación.

1. Preparación de tumorspheres retinoblastoma <ol><li> Obtener una muestra del tumor retinoblastoma en un protocolo aprobado por el IRB. </li><li> Prepare frescos define los medios de comunicación de células madre (Neurobasal-A los medios de comunicación, 1X suplemento de B-27 menos de vitamina A, 1X no esenciales solución de aminoácidos, 1X L-glutamina, 20 ng / mL FEAG, 10 ng / mL bFGF). </li><li> Mecánicamente desglosar el tejido del tumor con un bisturí con una técnica transversal de un tejido estéril, la cultura tratados con 6-así placa. Inmediatamente añadir 100 ml de definir los medios de comunicación de las células madre en el tejido suave y difusión del tejido picado con el bisturí. Añadir 5 ml de definir los medios de comunicación de las células madre para el bienestar. </li><li> Se incuba a 37 ° C, 5% de CO 2 en un incubador humidificado e inspeccionar todos los días. Algunos tumorspheres debe surgir a partir del tejido del tumor interrumpe casi de inmediato, con un número creciente en el transcurso de 2-4 días. </li><li> Semanales, tumorspheres centrifugar a 300xg durante 5 minutos, volver a suspender en recién preparada define los medios de comunicación de las células madre, y la pipeta hacia arriba y abajo 10 veces para interrumpir el más grande, las esferas central necrótica y permitir que nuevos ámbitos, sana a la forma. </li></ol> 2. Preparación de las células tumorales para la inyección <ol><li> Si se inyectan tumorspheres cultivadas, con cuidado las esferas de la pipeta en un tubo cónico de 15 ml. Pipeta suavemente hacia arriba y abajo con una pipeta serológica 10-15 veces para interrumpir las esferas. </li><li> Contar las células que se inyecta con una hemocitómetro, y alícuota al menos 50.000 células por inyección en un nuevo tubo. Prepárese para las inyecciones como sea posible en función del número de células disponibles. </li><li> Centrifugar a 300xg durante 5 minutos. Con cuidado, aspirar el sobrenadante y resuspender en 5 ml de PBS. Repetir la centrifugación y la aspiración, y resuspender en 10 l de PBS por inyección. </li></ol> 3. Preparación de los animales <ol><li> Utilizando una jeringa de insulina, administrar una inyección intraperitoneal de cóctel roedores anestesia (ketamina 37,6 mg / ml, la xilazina 1,92 mg / mL, acepromacina 0,38 mg / ml) a un l por gramo de peso corporal (GBW). Espere 5 minutos para que el animal se sedado. Comprobar el latido del corazón y el lugar del animal en un cojín de calefacción. Mantener los animales en un cojín de calefacción en todo momento hasta su recuperación. </li><li> Después de la sedación, administrar una gota de HCl proparacaína en el ojo derecho. Vuelva a colocar los animales en el teclado se calienta y esperar 1 minuto. </li><li> Administrar una gota de fenilefrina HCl en el ojo derecho. Vuelva a colocar el animal en el teclado se calienta y espere 5 minutos. Si la dilatación de la pupila no ha ocurrido, volver a administrar una gota y esperar otros 5 minutos. </li><li> Observar a los animales para detectar signos de movimiento o contracción muscular, que puede ocurrir en&gt; 30 minutos después de la sedación. Ante los primeros síntomas de movimiento, administrar 1 l / gbw de ketamina HCl (diluida a 10 mg / ml en PBS) y espere 5 minutos. </li></ol> 4. La inyección de células tumorales <ol><li> Coloque el animal anestesiado bajo el microscopio de su lado, con el ojo derecho hacia arriba y con la cabeza apoyada sobre una gasa y se centra en obtener un reflejo rojo en el ojo derecho del fondo de ojo (pupila debe ser dilatada en este momento) cuando se observa a través del microscopio . </li><li> Sostener el globo ocular derecho presionando suavemente al mismo tiempo con dos dedos sobre los párpados y mantener esta posición de manera constante durante el resto del procedimiento. </li><li> Utilizando una aguja estéril de calibre 30 aguja conectada a una jeringa Luer-lock, perforar el globo lateralmente a través de la conjuntiva y la esclera adyacente al limbo corneal en el área de la pars plana (Figura 1) y sólo a través de la coroides en la cavidad vítrea. Retire la aguja de la apertura. </li><li> Esterilizar la aguja Hamilton con un algodón con alcohol. Dibuja la suspensión de células en la jeringa Hamilton e inserte la aguja en el orificio hasta que la aguja se visualiza a través del microscopio detrás de la cámara en el vítreo, cerca de la retina. Con la ayuda de una segunda persona mientras se mantiene la aguja de manera constante en esta posición, presione el émbolo lenta. Retire la aguja. Si es necesario, use un hisopo de algodón para absorber cualquier líquido de la apertura. </li><li> Suavemente liberar la presión de los párpados para cerrar los párpados del ratón. </li><li> Administrar una gota de hipromelosa para cada ojo y devolver los animales al cojín calentado para la recuperación. </li></ol> 5. Seguimiento de los ratones inyectados y la cosecha de los tumores <ol><li> Todos los días, examinará los ojos inyectados de leucocoria (reflejo blanco papilar) y / o distensión periocular que por lo general se manifiesta 2-4 semanas después de la inyección. </li><li> Una vez que el crecimiento del tumor se detecta, la eutanasia de animales de acuerdo con las directrices institucionales. </li><li> Si el ojo y el tumor está cubierto por los párpados, el uso del bisturí para realizar dos incisiones ortogonal a la hendidura palpebral y levantar los colgajos de piel. </li><li> Bajo visualización con el microscopio estereoscópico, hacer una incisión circunferencial en el limbo, la eliminación de la córnea y el cristalino, y con mucho cuidado diseccionar la mayor parte de la masa del tumor de los tejidos con otros tweezers. Lugar del tumor en estéril RPMI-1640. </li><li> Utilizar pinzas para extraer lentamente el mundo abierto de la toma. Para asegurar un nervio óptico adjunto para la invasión del tumor se pueden visualizar, tire del mundo hasta que el nervio está expuesto y el uso de tijeras para cortar el nervio mayor tiempo posible. Lugar en formol al 10% para el procesamiento normal para el examen histológico. </li></ol> 6. Los resultados representativos: Tumorspheres retinoblastoma comenzarán a aparecer a partir del tejido desglosados ​​casi de inmediato, ya que se liberan de la masa tumoral. Dentro de 2-4 días, más tumorspheres se empiezan a formar y aumentar de tamaño. Las esferas tienden a ser regulares y presentan una estrategia bien definida, la membrana que rodea el conjunto secundario (Figura 2). El animal se presenta típicamente con leucocoria en las 4 semanas después de la inyección (Figura 3b), seguido por la ampliación de la tierra y la distensión de los tejidos circundantes 5-8 semanas después de la inyección al crecer el tumor (Figura 3c). <img src="/files/ftp_upload/2644/2644fig1.jpg" alt="Figura 1"/> Figura 1. Transversal diagrama del ojo del ratón destacando características mencionadas en el protocolo. <img src="/files/ftp_upload/2644/2644fig2.jpg" alt="Figura 2"/> Figura 2. Cultivo de células humanas in vitro retinoblastoma imágenes en un 4x), y b) aumento del objetivo de 10x. Células de retinoblastoma tumor primario producir tumorspheres con una forma regular esferoide y una membrana externa crujiente. Las barras de escala representan a) 500 m, y b) micras 200. <img src="/files/ftp_upload/2644/2644fig3.jpg" alt="Figura 3"/> Figura 3 Ojo de Rag2 -. / - Ratón que muestra una serie de características) normal, b) indicativo de una masa tumoral en la cavidad vítrea leucocoria, y c) una gran masa tumoral de llenar el mundo con distensión periocular asociada, hemorragia intraocular.

<ol>
	<li>Ch&#232;vez-Barrios, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+and+Nonmetastatic+Models+of+Retinoblastoma.">Metastatic and Nonmetastatic Models of Retinoblastoma.</a> Am. J. Pathol. 157, 1405-1412 (2000).</li><li>Suber, M. L., Hurwitz, M. Y., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+consequences+of+intraocular+administration+of+modified+adenoviral+vectors.">Immune consequences of intraocular administration of modified adenoviral vectors.</a> Hum. Gene Ther. 12, 833-838 (2001).</li><li>Mallam, J. N., Hurwitz, M. Y., Mahoney, T., Ch&#232;vez-Barrios, P., Hurwitz, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Gene+Transfer+into+Retinal+Cells+Using+Adenoviral+Vectors:+Dependence+on+Receptor+Expression.">Efficient Gene Transfer into Retinal Cells Using Adenoviral Vectors: Dependence on Receptor Expression.</a> Invest. Ophthalmol. Vis. Sci. 45, 1680-1687 (2004).</li></ol>

Los experimentos con animales se realizaron de acuerdo con las directrices y normas establecidas por el Colegio Baylor de Medicina IACUC Comité.

La técnica descrita en este documento facilita la propagación de los tumores de retinoblastoma intraocular en su entorno, intravítrea. La técnica de inyección intraocular se ha utilizado en el pasado para crear tumores de líneas celulares derivadas retinoblastoma-1, así como para entregar los vectores virales para la terapia génica 2,3 intraocular. Esta técnica ha sido utilizada con éxito para producir tumores humanos retinoblastoma mediante la inyección de las células del tumor primario y la inyección de tumorspheres así como la propagación de serie de los tumores de xenoinjerto. Evidencia visible de la formación de tumores (por lo general leucocoria) se nota por primera vez en 4 semanas, después de que la hemorragia intraocular y la distensión del globo y / o tejidos que rodean la órbita se desarrollan dentro de 5-8 semanas. Una minoría de los ratones inyectados dañar el ojo inyectado, provocando el cierre permanente de los párpados. En estos casos, la distensión es la única señal de la formación de tumores. Establecimiento de tumores murinos xenoinjertos no ha tenido éxito con todos los tumores de retinoblastoma humanos, a pesar de la propagación de los tumores de xenoinjerto establecido es de gran éxito. Esta observación sugiere que ciertas características del tumor primario, como la agresividad y el nivel de diferenciación o de algún otro factor desconocido, pueden estar influyendo en la capacidad de estos tumores a persistir en el ambiente ocular murino. La cantidad de tejido que pueden ser adquiridos a partir de un tumor retinoblastoma humanos es bastante pequeña, y hay importantes limitaciones en la capacidad de las células de cultivo retinoblastoma humanos in vitro, tales como tiempo de vida limitado, la pérdida de la histología del tumor sólido y cambios en el fenotipo celular. Este protocolo proporciona una forma relativamente sencilla de propagar el tumor humano y establecer un modelo murino de la enfermedad humana. Esto permite que más in vitro y la experimentación in vivo de la biología de retinoblastoma y ya el mantenimiento de la parte exterior del tumor del paciente.

<table border="1"><tbody><tr><th> Nombre </th><th> Empresa </th><th> Catálogo # </th><th> Comentarios </th></tr><tr><td> Clorhidrato de fenilefrina al 2,5% </td><td> Bausch &amp; Lomb </td><td> 053-11 </td><td> Oftálmica solución </td></tr><tr><td> 30 ga la aguja </td><td> BD </td><td> 305128 </td><td> Bisel regular </td></tr><tr><td> 10 ml Luer-Lock Jeringa </td><td> BD </td><td> 309604 </td><td></td></tr><tr><td> 3/10-cc jeringa de insulina </td><td> BD </td><td> 328431 </td><td></td></tr><tr><td> Torundas con alcohol </td><td> BD </td><td> 326895 </td><td></td></tr><tr><td> 6-Bueno Plate, el cultivo de tejidos tratados </td><td> BD </td><td> 353046 </td><td></td></tr><tr><td> Proparacaína HCl 0,5% </td><td> Mayordomo de la peste equina </td><td> 017239 </td><td> Oftálmica solución </td></tr><tr><td> La ketamina HCl 100mg/ml </td><td> Fort Dodge </td><td> 4402A </td><td></td></tr><tr><td> 10-l Hamilton jeringa </td><td> Hamilton Company </td><td> 7648-01 </td><td></td></tr><tr><td> 32 ga Hamilton Aguja </td><td> Hamilton Company </td><td> 7803-04 </td><td> Costumbre longitud - 0.5 &quot; </td></tr><tr><td> Neurobasal-A los medios de comunicación </td><td> Invitrogen </td><td> 10888-022 </td><td></td></tr><tr><td> B-27 Suplemento Menos vitamina A, 50 veces </td><td> Invitrogen </td><td> 12587-010 </td><td></td></tr><tr><td> RPMI-1640 </td><td> Mediatech </td><td> 10 a 040 CV </td><td></td></tr><tr><td> No esenciales solución de aminoácidos, 100X </td><td> Mediatech </td><td> 25 a 025-CI </td><td></td></tr><tr><td> L-Glutamina, 100X </td><td> Mediatech </td><td> 25 a 005-CI </td><td></td></tr><tr><td> Desechables bisturí # 11 </td><td> Miltex </td><td> 4-411 </td><td></td></tr><tr><td> Roedores Combo anestesia </td><td> n / a </td><td> n / a </td><td> En casa de farmacia formulación (ketamina 37,6 mg / ml, la xilazina 1,92 mg / mL, acepromacina 0,38 mg / mL) </td></tr><tr><td> Recombinante factor de crecimiento epidérmico (EGF) </td><td> StemCell Tecnologías </td><td> 02633 </td><td> Reconstituir a 10 mg / ml de solución de archivo </td></tr><tr><td> Recombinante humano del factor de crecimiento de fibroblastos básico (bFGF) </td><td> StemCell Tecnologías </td><td> 02634 </td><td> Reconstituir a 10 mg / ml de solución de archivo </td></tr><tr><td> OMS-75 Microscopio operaciones </td><td> Topcon Medical Systems </td><td> OMS-75 </td><td> Este modelo ha sido descontinuado </td></tr><tr><td> Formalina al 10% </td><td> VWR </td><td> 95042-908 </td><td></td></tr></tbody></table>

establishment and propagation of human retinoblastoma tumors in immune deficient mice

El cultivo de las células del tumor retinoblastoma en los medios de comunicación definidos de células madre da lugar a tumorspheres primaria que pueden ser cultivadas y se mantiene durante un tiempo limitado. Estos tumorspheres cultivadas pueden presentar fenotipos celulares muy diferentes en comparación con los tumores originales. Demostración de que las células cultivadas tienen la capacidad de formar nuevos tumores es importante asegurarse de que el modelo de las células cultivadas en la biología del tumor original. Aquí se presenta un protocolo para la propagación de los tumores humanos in vivo con retinoblastoma Rag2 - / - ratones deficientes inmunes. Cultivadas tumorspheres retinoblastoma humanos de paso bajo o de células obtenidas de recién cosechado tumores retinoblastoma humano se inyecta directamente en la cavidad vítrea de los ojos de formar tumores murinos en 2-4 semanas. Estos tumores pueden ser cosechadas y sea más pasajes en los ojos de murino in vivo o que han crecido como tumorspheres in vitro. Propagación se ha realizado con éxito durante al menos tres pasajes, estableciendo así una fuente continua de tejidos humanos para la experimentación retinoblastoma más. Wesley S. Bond y Wadhwa Lalita son co-autores de primera.

Watch this Scientific Journal Video about Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice at JoVE.com

Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Se describe un método para propagar los tumores humanos en ratones retinoblastoma. Las células tumorales se inyectan directamente en los ojos de ratones inmunes deficientes. Los tumores secundarios se han establecido con éxito utilizando las células cosechadas directamente de tumores humanos y tumorspheres cultivadas.

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 ...

establishment-propagation-human-retinoblastoma-tumors-immune

Research

JoVE Journal

Medicine

11.7K vistas.  Baylor College of Medicine. Se describe un método para propagar los tumores humanos en ratones retinoblastoma. Las células tumorales se inyectan directamente en los ojos de ratones inmunes deficientes. Los tumores secundarios se han establecido con éxito utilizando las células cosechadas directamente de tumores humanos y tumorspheres cultivadas.

Video: Establishment and Propagation of Human Retinoblastoma Tumors in Immune Deficient Mice

Flexible Colonoscopy in Mice to Evaluate the Severity of Colitis and Colorectal Tumors Using a Validated Endoscopic Scoring System

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental endoscopy has been highly useful for studies that require repeated assessments in a single laboratory animal, such as those evaluating mechanisms of chronic inflammatory bowel disease and the progression of colorectal cancer. However, the methods used across studies are highly variable. At least three endoscopic scoring systems have been published for murine colitis and published protocols for the assessment of colorectal tumors fail to address the presence of concomitant colonic inflammation. This study develops and validates a reproducible endoscopic scoring system that integrates evaluation of both inflammation and tumors simultaneously. This novel scoring system has three major components: 1) assessment of the extent and severity of colorectal inflammation (based on perianal findings, transparency of the wall, mucosal bleeding, and focal lesions), 2) quantitative recording of tumor lesions (grid map and bar graph), and 3) numerical sorting of clinical cases by their pathological and research relevance based on decimal units with assigned categories of observed lesions and endoscopic complications (decimal identifiers). The video and manuscript presented herein were prepared, following IACUC-approved protocols, to allow investigators to score their own experimental mice using a well-validated and highly reproducible endoscopic methodology, with the system option to differentiate distal from proximal endoscopic colitis (D-PECS).

Over the past few years, new generation endoscopes have emerged as important diagnostic research aids for evaluating murine colitis and colorectal tumors. We present herein a detailed protocol for endoscopic assessment of inflammation and colorectal tumors in mice, as well as a novel scoring system that uses decimal identifiers to document the endoscopic severity of colitis and colorectal tumors.

La colonoscopia flexible en ratones para evaluar la gravedad de la colitis y colorrectal tumores utilizando un sistema validado de puntuación endoscópica

The use of modern endoscopy for research purposes has greatly facilitated our understanding of gastrointestinal pathologies. In particular, experimental ...

Investigación

Medicina

Induction of Invasive Transitional Cell Bladder Carcinoma in Immune Intact Human MUC1 Transgenic Mice:  A Model for Immunotherapy Development

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy and/or cytotoxic chemotherapy. To induce bladder cancer, C57BL/6 mice (MUC1.Tg and wild type) were treated orally with the carcinogen N-butyl-N-(4-hydroxybutyl)nitrosamine (OH-BBN) at 3.0 mg/day, 5 days/week for 12 weeks. To assess the effects of OH-BBN on serum cytokine profile during tumor development, whole blood was collected via submandibular bleeds prior to treatment and every four weeks. In addition, a MUC1-targeted peptide vaccine and placebo were administered to groups of mice weekly for eight weeks. Multiplex fluorometric microbead immunoanalyses of serum cytokines during tumor development and following vaccination were performed. At termination, interferon gamma (IFN-&#947;)/interleukin-4 (IL-4) ELISpot analysis for MUC1 specific T-cell immune response and histopathological evaluations of tumor type and grade were performed. The results showed that: (1) the incidence of bladder cancer in both MUC1.Tg and wild type mice was 67%; (2) transitional cell carcinomas (TCC) developed at a 2:1 ratio compared to squamous cell carcinomas (SCC); (3) inflammatory cytokines increased with time during tumor development; and (4) administration of the peptide vaccine induces a Th1-polarized serum cytokine profile and a MUC1 specific T-cell response. All tumors in MUC1.Tg mice were positive for MUC1 expression, and half of all tumors in MUC1.Tg and wild type mice were invasive. In conclusion, using a team approach through the coordination of the efforts of pharmacologists, immunologists, pathologists and molecular biologists, we have developed an immune intact transgenic mouse model of bladder cancer that expresses hMUC1.

An N-butyl-N-(4-hydroxybutyl)nitrosamine-induced bladder cancer model was developed in human mucin 1 (MUC1) transgenic mice for the purpose of testing MUC1-directed immunotherapy. After administering a MUC1-targeted peptide vaccine, a cytotoxic T lymphocyte response to MUC1 was confirmed by measuring serum cytokine levels and T-cell specific activity.

La inducción de la transición de vejiga invasivo Carcinoma en inmune intacto humanos MUC1 Ratones Transgénicos: Un Modelo para el Desarrollo Inmunoterapia

A preclinical model of invasive bladder cancer was developed in human mucin 1 (MUC1) transgenic (MUC1.Tg) mice for the purpose of evaluating immunotherapy ...

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent marker. Mouse models (brain tumor and arthritis) were used to evaluate the usefulness of this method. Saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles tagged with CellVue Maroon (CVM) fluorophore were administered intravenously. Animals were then placed in the rotational holder (MARS) of the in vivo imaging system. Images were acquired in 10&#176; steps over 380&#176;. A rectangular&#160;region of interest (ROI) was placed across the full image width at the model disease site. Within the ROI, and for every image, mean fluorescence intensity was computed after background subtraction. In the mouse models studied, the labeled nanovesicles were taken up in both the orthotopic and transgenic brain tumors, and in the arthritic sites (toes and ankles). Curve analysis of the multi angle image ROIs determined the angle with the highest signal. Thus, the optimal angle for imaging each disease site was characterized. The MAROI method applied to imaging of fluorescent compounds is a noninvasive, economical, and precise tool for in vivo quantitative analysis of the disease states in the described mouse models.

In Vivo Optical Imaging of Brain Tumors and Arthritis Using Fluorescent SapC-DOPS Nanovesicles

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo quantitation of a fluorescent marker delivered by saposin C (SapC)-dioleoylphosphatidylserine (DOPS) nanovesicles. Employing mouse models of cancer and arthritis, we demonstrate how the MAROI signal curve analysis can be used for the precise mapping and biological characterization of disease processes.

En vivo de imágenes óptico de tumores del cerebro y la artritis Usando fluorescente SAPC-DOPS nanovesículas

We describe a multi-angle rotational optical imaging (MAROI) system for in vivo monitoring of physiopathological processes labeled with a fluorescent ...

Femoral Bone Marrow Aspiration in Live Mice

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed step-by-step technical procedure for an analogous procedure in live mice which allows for serial characterization of cells present in the BM. This procedure facilitates studies aimed to detect the presence of exogenously administered cells within the BM of mice as would be done in xenograft studies for instance. Moreover, this procedure allows for the retrieval and characterization of cells enriched in the BM such as hematopoietic stem and progenitor cells (HSPCs) without sacrifice of mice. Given that the cellular composition of peripheral blood is not necessarily reflective of proportions and types of stem and progenitor cells present in the marrow, procedures which provide access to this compartment without requiring termination of the mice are very helpful. The use of femoral bone marrow aspiration is illustrated here for cytological analysis of marrow cells, flow cytometric characterization of the hematopoietic stem/progenitor compartment, and culture of sorted HSPCs obtained by femoral BM aspiration compared with conventional marrow harvest.

This protocol describes a procedure for serial sampling of femoral bone marrow (BM) without requiring the sacrifice of mice. This procedure facilitates longitudinal studies of the BM composition of mice over time and provides serial access to cells within the BM for ex vivo and transplantation studies.

Femoral Aspiración de médula ósea en ratones vivos

Serial sampling of the cellular composition of bone marrow (BM) is a routine procedure critical to clinical hematology. This protocol describes a detailed ...

Creating Anatomically Accurate and Reproducible Intracranial Xenografts of Human Brain Tumors

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live animal &#8211;&#160;particularly in sites with unique physiological and architectural qualities such as the brain. In vitro and ectopic models cannot account for features such as vasculature, blood brain barrier, metabolism, drug delivery and toxicity, and a host of other relevant factors. Orthotopic models have their limitations too, but with proper technique tumor cells of interest can be accurately engrafted into tissue that most closely mimics conditions in the human brain. By employing methods that deliver precisely measured volumes to accurately defined locations at a consistent rate and pressure, mouse models of human brain tumors with predictable growth rates can be reproducibly created and are suitable for reliable analysis of various interventions. The protocol described here focuses on the technical details of designing and preparing for an intracranial injection, performing the surgery, and ensuring successful and reproducible tumor growth and provides starting points for a variety of conditions that can be customized for a range of different brain tumor models.

The brain is a unique site with qualities that are not well represented by in vitro or ectopic analyses. Orthotopic mouse models with reproducible location and growth characteristics can be reliably created with intracranial injections using a stereotaxic fixation instrument and a low pressure syringe pump.

Crear anatómicamente exactos y reproducibles intracraneal xenoinjertos de tumores cerebrales humanos

Orthotopic tumor models are currently the best way to study the characteristics of a tumor type, with and without intervention, in the context of a live ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

Establecimiento de Human epitelial Enteroids y Colonoids de Whole Tejidos y Biopsia

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Establishment of a Human Multiple Myeloma Xenograft Model in the Chicken to Study Tumor Growth, Invasion and Angiogenesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the lack of the bone microenvironment and auto/paracrine growth factors human MM cells are difficult to cultivate. Therefore, there is an urgent need to establish proper in vitro and in vivo culture systems to study the action of novel therapeutics on human MM cells. Here we present a model to grow human multiple myeloma cells in a complex 3D environment in vitro and in vivo. MM cell lines OPM-2 and RPMI-8226 were transfected to express the transgene GFP and were cultivated in the presence of human mesenchymal cells and collagen type-I matrix as three-dimensional spheroids. In addition, spheroids were grafted on the chorioallantoic membrane (CAM) of chicken embryos and tumor growth was monitored by stereo fluorescence microscopy. Both models allow the study of novel therapeutic drugs in a complex 3D environment and the quantification of the tumor cell mass after homogenization of grafts in a transgene-specific GFP-ELISA. Moreover, angiogenic responses of the host and invasion of tumor cells into the subjacent host tissue can be monitored daily by a stereo microscope and analyzed by immunohistochemical staining against human tumor cells (Ki-67, CD138, Vimentin) or host mural cells covering blood vessels (desmin/ASMA).
In conclusion, the onplant system allows studying MM cell growth and angiogenesis in a complex 3D environment and enables screening for novel therapeutic compounds targeting survival and proliferation of MM cells.

Human multiple myeloma (MM) cells require the supportive microenvironment of mesenchymal cells and extracellular matrix components for survival and proliferation. We established an in vivo chicken embryo model with engrafted human myeloma and mesenchymal cells to study effects of cancer drugs on tumor growth, invasion and angiogenesis.

Establecimiento de un modelo de xenoinjerto Humano mieloma múltiple en el pollo para estudiar el crecimiento del tumor, la invasión y la angiogénesis

Multiple myeloma (MM), a malignant plasma cell disease, remains incurable and novel drugs are required to improve the prognosis of patients. Due to the ...

Induction of Accelerated Atherosclerosis in Mice: The "Wire-Injury" Model

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. Moreover, by rupture of the defective vascular wall, atherosclerosis induces occlusive thrombus formation, which represents the main cause of myocardial infarction or stroke and the most frequent cause of death. Despite the advances in the cardiovascular field, many questions remain unanswered, and additional basic research is essential to improve our understanding of the molecular mechanisms during atherosclerosis and its effects. Due to limited clinical studies, there is a need for representative animal models recreating atherosclerotic conditions such as neointima formation after stent implantation, balloon angioplasty, or endarterectomy. Since the mouse presents many advantages and is the most frequently used model for studying molecular processes, the current study proposes an invasive procedure of endothelial denudation, also known as the wire-injury model, which is representative of the human condition of neointima formation in arteries after revascularization procedures.

Induction of Accelerated Atherosclerosis in Mice: The &quot;Wire-Injury&quot; Model

This study describes an invasive procedure for the induction of accelerated atherosclerosis in mice. In comparison to other methods using electric- or cryo-induced injury, mechanical-induced injury mimics the human condition of restenosis after revascularization therapies and is ideal for the study of the molecular mechanisms involved.

Inducción de la Aterosclerosis Acelerada en Ratones: El Modelo de "Lesiones por Alambre"

Atherosclerosis is a proliferative fibro-inflammatory disease developing in the arterial wall, inducing a deficient blood flow or a lack of blood flow. ...

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

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

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

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

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

Induction of Atherosclerotic Plaques Through Activation of Mineralocorticoid Receptors in Apolipoprotein E-deficient Mice

Atherosclerosis is due to a chronic inflammatory response affecting vascular endothelium and is promoted by several factors such as hypertension, dyslipidemia, and diabetes. To date, there is evidence to support a role for circulating aldosterone as a risk factor for the development of cardiovascular disease. Transgenic mouse models have been generated to study cellular and molecular processes leading to atherosclerosis. In this manuscript, we describe a protocol that takes advantage of continuous infusion of aldosterone in ApoE-/- mice and generates atherosclerotic plaques in the aortic root after 4 weeks of treatment. We, therefore, illustrate a method for quantification and characterization of atherosclerotic lesions at the aortic root level. The added value of aldosterone infusion is represented by the generation of atherosclerotic lesions rich in lipid and inflammatory cells after 4 weeks of treatment. We describe in detail the staining procedures to quantify lipid and macrophage content within the plaque. Notably, in this protocol, we perform heart tissue-embedding in OCT in order to preserve the antigenicity of cardiac tissue and facilitate detectability of antigens of interest. Analysis of the plaque phenotype represents a valid approach to study the pathophysiology of atherosclerosis development and to identify novel pharmacological targets for the development of anti-atherogenic drugs.

We describe a protocol to induce atherosclerosis in the aortic root of ApoE-/- mice fed with an atherogenic diet, through a continuous release of aldosterone. Methods to characterize plaque composition are also described.

Inducción de las placas ateroscleróticas a través de la activación de los receptores de mineralocorticoides en ratones deficientes en apolipoproteína E