Esta investigación fue financiada por el Estado de Bankhead-Coley Equipo Científico Grant Florida (2BT03), los Institutos Nacionales de / Instituto Nacional del Cáncer de la Salud (1R21CA164322-01) y el Equipo de Ciencia de subvención de Moffitt Cancer Center. Este trabajo ha sido financiado en parte por el Fondo para la Investigación Traslacional Core en el H. Lee Centro de Investigación del Cáncer Moffitt Instituto, un Centro Integral del Cáncer del NCI señalado (P30-CA076292). El acceso a las células primarias se hizo posible a través del Protocolo de Cuidado Total del Cáncer en el Centro del Cáncer Moffitt.

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	<li>Salmon, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+differential+sensitivity+of+human-tumor+stem+cells+to+anticancer+drugs.">Quantitation of differential sensitivity of human-tumor stem cells to anticancer drugs.</a> N Engl J Med. 298, 1321-1327 (1978).</li><li>Suggitt, M., Bibby, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=50+years+of+preclinical+anticancer+drug+screening:+empirical+to+target-driven+approaches.">50 years of preclinical anticancer drug screening: empirical to target-driven approaches.</a> Clin Cancer Res. 11, 971-981 (2005).</li><li>Kirshner, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unique+three-dimensional+model+for+evaluating+the+impact+of+therapy+on+multiple+myeloma.">A unique three-dimensional model for evaluating the impact of therapy on multiple myeloma.</a> Blood. 112, 2935-2945 (2008).</li><li>Durie, B. G., Jacobson, J., Barlogie, B., Crowley, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnitude+of+response+with+myeloma+frontline+therapy+does+not+predict+outcome:+importance+of+time+to+progression+in+southwest+oncology+group+chemotherapy+trials.">Magnitude of response with myeloma frontline therapy does not predict outcome: importance of time to progression in southwest oncology group chemotherapy trials.</a> J Clin Oncol. 22, 1857-1863 (2004).</li><li>Harousseau, J. L., Attal, M., Avet-Loiseau, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+complete+response+in+multiple+myeloma.">The role of complete response in multiple myeloma.</a> Blood. 114, 3139-3146 (2009).</li><li>Khin, Z. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+preclinical+assay+for+chemosensitivity+in+multiple+myeloma.">A preclinical assay for chemosensitivity in multiple myeloma.</a> Cancer Res. 74, 56-67 (2014).</li><li>Misund, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+measurement+of+drug+sensitivity+of+myeloma+cells+co-cultured+with+bone+marrow+stromal+cells.">A method for measurement of drug sensitivity of myeloma cells co-cultured with bone marrow stromal cells.</a> J Biomol Screen. 18, 637-646 (2013).</li><li>Ramasamy, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescence-based+experimental+model+to+evaluate+the+concomitant+effect+of+drugs+on+the+tumour+microenvironment+and+cancer+cells.">Fluorescence-based experimental model to evaluate the concomitant effect of drugs on the tumour microenvironment and cancer cells.</a> Br J Haematol. 157, 564-579 (2012).</li><li>McMillin, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+cell-specific+bioluminescence+platform+to+identify+stroma-induced+changes+to+anticancer+drug+activity.">Tumor cell-specific bioluminescence platform to identify stroma-induced changes to anticancer drug activity.</a> Nat Med. 16, 483-489 (2010).</li><li>Koh, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+cells+for+microscopy+using+cytospin.">Preparation of cells for microscopy using cytospin.</a> Methods Enzymol. 533, 235-240 (2013).</li><li>Al-Quran, S. Z., Yang, L., Magill, J. M., Braylan, R. C., Douglas-Nikitin, V. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessment+of+bone+marrow+plasma+cell+infiltrates+in+multiple+myeloma:+the+added+value+of+CD138+immunohistochemistry.">Assessment of bone marrow plasma cell infiltrates in multiple myeloma: the added value of CD138 immunohistochemistry.</a> Hum Pathol. 38, 1779-1787 (2007).</li><li>Najar, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stromal+cells+promote+or+suppress+the+proliferation+of+T+lymphocytes+from+cord+blood+and+peripheral+blood:+the+importance+of+low+cell+ratio+and+role+of+interleukin-6.">Mesenchymal stromal cells promote or suppress the proliferation of T lymphocytes from cord blood and peripheral blood: the importance of low cell ratio and role of interleukin-6.</a> Cytotherapy. 11, 570-583 (2009).</li><li>Scott, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phase+i+trialist.">Phase i trialist.</a> Lancet Oncol. 13, 236 (2012).</li></ol>

The authors have no conflict of interest to disclose.

En resumen, este es un método potente rendimiento y alta para cuantificar la quimiosensibilidad de las células MM primarias y farmacodinámica ex vivo de fármacos en una reconstrucción de la médula ósea. Los pasos críticos dentro de este protocolo son la siembra adecuada de los pozos y centrándose adecuada (véase la Figura 2 para los resultados esperados): asegurar que las células estromales MM y se distribuyen de manera uniforme, que las células estromales son adherentes a la parte ...

El objetivo de este método es caracterizar la sensibilidad a los fármacos de mieloma múltiple (MM) células primarias a un panel de agentes ex vivo, lo más cerca posible a las condiciones fisiológicas, y con suficiente precisión que estos resultados se pueden utilizar para identificar quimiorresistente sub- poblaciones dentro de la carga tumoral y, en última instancia, para parametrizar modelos computacionales diseñados para estimar la respuesta clínica. Los modelos computacionales son herramientas poderosas para analizar sistemas complejos, como las interacciones huésped-terapia del cáncer. Sin embargo, los modelos sólo son tan buenos como los datos utilizados para parametrizar ellos. Desafortunadamente, la mayoría de los datos disponibles en la literatura no se pueden utilizar en su forma actual para parametrizar tales modelos, ya que a menudo se obtienen en condiciones experimentales mutuamente excluyentes. De este modo, los nuevos experimentos se requieren a menudo con el objetivo de obtener el conjunto de parámetros experimentales necesarios. La mayoría de los ensayos de viabilidad, sin embargo, son destructivas y THnos limitamos a un pequeño número de puntos de tiempo, a menudo sólo uno. Esto perjudica en gran medida el uso de ensayos chemosensitivity parametrizar modelos computacionales, ya que tales experimentos no proporcionan información sobre la dinámica temporal del sistema. Esto es especialmente cierto con las células de cáncer primario, que a menudo se limitan a unos pocos millones por muestra, y tienen una vida útil corta después de la biopsia, lo que limita el número de condiciones experimentales disponibles. Además, la mayoría de los ensayos de viabilidad requieren la separación del cáncer de las células no cancerosas (estroma) en el momento de la cuantificación, lo que añade trabajo extra para el protocolo, perturba aún más las células, y limita el número de experimentos que puede realizarse . Hace 40 años, salmón y colaboradores 1 propusieron su famoso ensayo de formación de colonias in vitro para la evaluación de la quimiosensibilidad de las células tumorales. Desafortunadamente este ensayo encontró un éxito desigual debido al pequeño número de múltiples myeloma (MM) muestras de pacientes que eran capaces de formar colonias bajo condiciones de control: Se estima que sólo un pequeño subgrupo de 0,001% a 0,1% de las células MM eran capaces de replicación, se denominan como células madre de mieloma múltiple 2. Esto limita significativamente la tasa de éxito del ensayo y el número de medicamentos que podrían ser probado en un tiempo, incluso en modelos más recientes 3. Este problema es mucho más importante ahora, cuando agentes MM (estándar o experimentales) son más numerosos que hace cuatro décadas. Una limitación importante de estos primeros ensayos es su producción dichotomized: ya sea un paciente es &quot;sensible&quot; o &quot;resistente&quot; a un medicamento en particular. No se proporciona información sobre el grado de sensibilidad y la heterogeneidad de la población tumor. Por lo tanto, los pacientes con pequeñas subpoblaciones de células de rápido crecimiento resistentes serían clasificados como &quot;sensibles&quot; a la terapia, sino que recaer en breve, por lo que no beneficio de tratamiento. Dada la importancia de la duración de la respuesta en la supervivencia global (OS) de pacientes con cáncer de 4,5, está claro cómo estos ensayos no fueron capaces de estimar correctamente OS consistentemente. Con el fin de evitar estas limitaciones, y ser capaz de generar modelos computacionales específicos del paciente que estimar la respuesta clínica personalizado a un grupo de medicamentos, hemos desarrollado un método para la sensibilidad de drogas prueba no destructiva de mieloma múltiple (MM) líneas celulares y las células MM primarias en una reconstrucción ex vivo del microambiente de la médula ósea, incluyendo la matriz extracelular y el estroma 6. Este ensayo, sin embargo, tenía la limitación de depender de una diapositiva de microfluidos comercial en particular, cuyas dimensiones y el coste restringido el número de experimentos o medicamentos que podrían llevarse a cabo a la vez en un equipo determinado. El sistema aquí descrito se extiende este ensayo original en un alto-throughput organotípicos plataforma de dosis-respuesta, para el cribado in vitro de fármacos, basado en un algoritmo de análisis de imagen digital para no destructiva cuantificar la viabilidad celular. Cada pocillo en 384 o 1536 pocillos placas es una reconstrucción 3D del microambiente de la médula ósea, incluyendo células primarias mm, matriz extracelular, y el estroma y factores de crecimiento derivados del paciente. Microscopía vivo y análisis digital de imágenes se utilizan para detectar eventos de muerte celular en diferentes concentraciones de fármaco, que se utilizan para generar superficies de dosis-respuesta. De los datos in vitro, un modelo matemático identifica el tamaño y la quimiosensibilidad de sub-poblaciones dentro de la carga tumoral del paciente, y se puede utilizar para simular cómo respondería el tumor al fármaco (s) en condiciones fisiológicas en un régimen de clínica 6 ( Figura 1). Las principales novedades de esta plataforma son: (a) pequeño número de células cancerosas requeridos (1.000-10.000 por concent drogasración); (B) la evaluación de la eficacia del fármaco en condiciones fisiológicas (matriz extracelular, estroma, factores de crecimiento derivados del paciente); (C) Ninguna toxicidad de los marcadores de viabilidad 7 ya que sólo de imágenes de campo claro se utiliza, por lo tanto no hay necesidad de transfectar células con fluorescencia o bioluminiscencia 8 9; (D) de formación de imágenes continua proporciona efecto de la droga como una función de la concentración y tiempo de exposición (farmacodinámica); y (e) la integración entre in vitro y modelos computacionales evolutivas, para estimar el resultado clínico 6 (Silva et al., en preparación). El factor clave que permite que el algoritmo de análisis de imagen digital para discernir el cáncer de células del estroma es su diferente afinidad a la parte inferior del pozo: células MM no se adhieren, y permanecen en suspensión en la matriz, mientras que las células estromales se adhieren a la parte inferior de la bien y luego estirar. Esta separación se produce a pesar de que MM y un estromare sembrado al mismo tiempo, y se produce durante el proceso de O / N de la incubación. El desacelerándose en la centrífuga después de la siembra de la placa se acelera aún más este proceso. Como consecuencia, las células MM aparecen en la imagen como discos redondos brillantes rodeadas por un anillo oscuro, mientras que el estroma mantiene un perfil bajo y un tono más oscuro (Figura 2). Por lo tanto, el ensayo en su forma actual es el más adecuado para las células cancerosas no adherentes, aunque los ajustes en el algoritmo se podría hacer con el cáncer independiente y estroma en base a otras características morfológicas, como el tamaño y forma. En este protocolo se describen dos tipos de matriz extracelular (colágeno y matriz de membrana basal), así como dos tipos de co-cultivos, con (células madre mesenquimales derivadas de médula ósea) BMSC y HUVECS, en representación de los nichos intersticiales y perivasculares de la médula ósea , respectivamente. El uso de una placa de 384 pocillos, 5 concentraciones por drogas y dos repeticiones, que fueron capaces de test hasta 31 fármacos diferentes con la muestra de un solo paciente. En placas de 1536 pocillos este número es 127. La Figura 3 representa la disposición utilizada actualmente para la siembra de células manual, mientras que la Figura Suplementaria 1 representa la distribución óptima usando una pipeta robótica. En el diseño de la figura 3, cada placa de 384 pocillos puede llevar a 3 muestras de pacientes con 7 fármacos diferentes, o una muestra de pacientes evaluados con 21 drogas. Cada fármaco se representa por 5 concentraciones, y cada condición se siembra en los duplicados. 4 pozos de control (ningún fármaco añadido) se siembran para cada conjunto de 7 medicamentos (por ejemplo, pozos 36-39, 126-129 y 200-203). Para garantizar la potencia de los medicamentos que se utilizan, una línea celular de mieloma humano (por ejemplo, H929 o MM1.S) también es cabeza de serie, y drogada por duplicado a la mayor concentración de cada uno de los fármacos utilizados (pozos 76-89). El control de la línea de células positivas se asegura de que los medicamentos que se utilizan tienen una potencia adecuada, ya que sus curvas de respuesta de dosis son comparables acexperimentos Ross. Dos pocillos se sembraron con una línea celular de mieloma como control para las condiciones ambientales, en otras palabras, para detectar posibles problemas en la incubadora de banco durante la exploración (pozos 75 y 90). La enumeración de los pozos sigue un patrón en zigzag con el fin de reducir la distancia recorrida por la etapa motorizada del microscopio, lo que reduce el tiempo de adquisición y reduce el desgaste del equipo.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>EVOS FL AUTO</td>
			<td>AMG</td>
			<td>AMAFD1000 + AMC1000</td>
			<td>Digital microscope equipped with motorized stage and bench top incubator.</td>
		</tr>
		<tr>
			<td>384-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3683 SIGMA</td>
			<td>Corning CellBIND 384 well plates 
			384 well plate, polystyrene, CellBIND surface, sterile, clear flat bottom, black, w/lid</td>
		</tr>
		<tr>
			<td>1,536-well plate CellBind</td>
			<td>CORNING</td>
			<td>CLS3832 ALDRICH</td>
			<td>Corning 1536 well plates, low base, surface treatment CellBIND, sterile</td>
		</tr>
		<tr>
			<td>Ficoll-Paque Plus&#160;</td>
			<td>GE Healthcare</td>
			<td>GE17-1440-02 SIGMA</td>
			<td>For in vitro isolation of lymphocytes from human peripheral blood.</td>
		</tr>
		<tr>
			<td>CD138 magnetic beads</td>
			<td>Miltenyi&#160;</td>
			<td>130-051-301</td>
			<td>Antibody conjugated magnetic beads for selection of CD138+ cells.</td>
		</tr>
		<tr>
			<td>LS columns</td>
			<td>Miltenyi&#160;</td>
			<td>&#160;130-042-401</td>
			<td>Column for separation of cells.</td>
		</tr>
		<tr>
			<td>MidiMACS Separator</td>
			<td>Miltenyi&#160;</td>
			<td>130-042-302</td>
			<td>Magnet for separation column.</td>
		</tr>
		<tr>
			<td>Matrigel</td>
			<td>Sigma-Aldrich</td>
			<td>E6909 SIGMA</td>
			<td>ECM Gel from Engelbreth-Holm-Swarm murine sarcoma</td>
		</tr>
		<tr>
			<td>Getrex</td>
			<td>Life Technologies</td>
			<td>A1413201</td>
			<td>LDEV-Free Reduced Growth Factor Basement Membrane Matrix</td>
		</tr>
		<tr>
			<td>HUVEC</td>
			<td>Life Technologies</td>
			<td>C-003-25P-B</td>
			<td>Human Umbilical Vein Endothelial Cells (HUVEC )</td>
		</tr>
		<tr>
			<td>RPMI-1640 media</td>
			<td>GIBCO</td>
			<td>11875-093</td>
			<td>RPMI 1640 Medium + L-Glutamine + Phenol Red</td>
		</tr>
		<tr>
			<td>FBS Heat inactivated</td>
			<td>GIBCO</td>
			<td>10082-147</td>
			<td>Fetal Bovine Serum, certified, heat inactivated, US origin</td>
		</tr>
		<tr>
			<td>3.1mg/mL Bovine collagen type I</td>
			<td>Advanced Biomatrix</td>
			<td>5005-B</td>
			<td>Bovine Collagen Solution, Type I, 3 mg/ml, 100 ml</td>
		</tr>
		<tr>
			<td>Precision XS</td>
			<td>BIOTEK</td>
			<td>PRC3841</td>
			<td>Robotic pipettor system</td>
		</tr>
	</tbody>
</table>

an organotypic high throughput system for characterization of drug sensitivity of primary multiple myeloma cells

En este trabajo se describe un nuevo método que combina ensayos ex vivo de sensibilidad de drogas y análisis de imágenes digitales para estimar quimiosensibilidad y heterogeneidad de mieloma múltiple las células derivadas del paciente (MM). Este enfoque consiste en la siembra de células MM primarias recién extraídos de aspirados de médula ósea en las cámaras de microfluidos implementadas en placas de múltiples pocillos, cada uno compuesto de una reconstrucción del microambiente de la médula ósea, incluyendo la matriz extracelular (colágeno de la membrana basal o matriz) y el estroma (el paciente células madre mesenquimales derivadas) o células endoteliales derivadas de seres humanos (HUVEC). Las cámaras están drogados con diferentes agentes y concentraciones, y se crean imágenes de forma secuencial durante 96 horas a través de la microscopía de campo claro, en un microscopio motorizado equipado con una cámara digital. Software de análisis de imagen digital detecta células vivas y muertas de la presencia o ausencia de movimiento de la membrana, y genera curvas de cambio en la viabilidad como una función of concentración del fármaco y el tiempo de exposición. Usamos un modelo computacional para determinar los parámetros de la quimiosensibilidad de la población tumor para cada fármaco, así como el número de sub-poblaciones presentes como una medida de la heterogeneidad tumoral. Estos modelos adaptados al paciente se pueden utilizar para simular los regímenes terapéuticos y estimar la respuesta clínica.

El flujo del experimento se describe brevemente en la Figura 1 Si todos los pasos se han completado con éxito, las imágenes obtenidas por el microscopio debe ser equivalente a la Figura 2:. Viven células MM deben verse claramente discos tan brillantes y BMSCs o HUVECs deben ser apenas visible y bien distribuida en el fondo. Como se ve en la Figura 2A, las células MM varían en tamaño de un paciente a otro, pero en general son más pequeños que las líneas celulare...

Watch this Scientific Journal Video about An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells at JoVE.com

An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Un Sistema organotípicos de alto rendimiento para la Caracterización de la sensibilidad a los fármacos de las células del mieloma múltiple primaria

In this work we describe a novel approach that combines ex vivo drug sensitivity assays and digital image analysis to estimate chemosensitivity and ...

an-organotypic-high-throughput-system-for-characterization-drug

Research

JoVE Journal

Medicine

8.5K vistas.  H. Lee Moffitt Cancer Center and Research Institute. We describe a high-throughput drug sensitivity assay for primary multiple myeloma cells. It consists of a reconstruction of the bone marrow microenvironment (including extracellular matrix and stroma) in multi-well plates, and a non-invasive method for longitudinal quantification of cell viability.

Video: An Organotypic High Throughput System for Characterization of Drug Sensitivity of Primary Multiple Myeloma Cells

Coculture System with an Organotypic Brain Slice and 3D Spheroid of Carcinoma Cells

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in particular the role of the resident (stromal) cells. Studies in primary carcinomas demonstrate the influence of the microenvironment on metastasis, even on prognosis1,2. Especially the tumor associated macrophages (TAM) support migration, invasion and proliferation3. Interestingly, the major target sites of metastasis possess tissue-specific macrophages, such as Kupffer cells in the liver or microglia in the CNS. Moreover, the metastatic sites also possess other tissue-specific cells, like astrocytes. Recently, astrocytes were demonstrated to foster proliferation and persistence of cancer cells4,5. Therefore, functions of these tissue-specific cell types seem to be very important in the process of brain metastasis6,7.
Despite these observations, however, up to now there is no suitable in vivo/in vitro model available to directly visualize glial reactions during cerebral metastasis formation, in particular by bright field microscopy. Recent in vivo live imaging of carcinoma cells demonstrated their cerebral colonization behavior8. However, this method is very laborious, costly and technically complex. In addition, these kinds of animal experiments are restricted to small series and come with a substantial stress for the animals (by implantation of the glass plate, injection of tumor cells, repetitive anaesthesia and long-term fixation). Furthermore, in vivo imaging is thus far limited to the visualization of the carcinoma cells, whereas interactions with resident cells have not yet been illustrated. Finally, investigations of human carcinoma cells within immunocompetent animals are impossible8.
For these reasons, we established a coculture system consisting of an organotypic mouse brain slice and epithelial cells embedded in matrigel (3D cell sphere). The 3D carcinoma cell spheres were placed directly next to the brain slice edge in order to investigate the invasion of the neighboring brain tissue. This enables us to visualize morphological changes and interactions between the glial cells and carcinoma cells by fluorescence and even by bright field microscopy. After the coculture experiment, the brain tissue or the 3D cell spheroids can be collected and used for further molecular analyses (e.g. qRT-PCR, IHC, or immunoblot) as well as for investigations by confocal microscopy. This method can be applied to monitor the events within a living brain tissue for days without deleterious effects to the brain slices. The model also allows selective suppression and replacement of resident cells by cells from a donor tissue to determine the distinct impact of a given genotype. Finally, the coculture model is a practicable alternative to in vivo approaches when testing targeted pharmacological manipulations.

The organotypic brain slice coculture with carcinoma cells enables visualizing morphological changes by fluorescence as well as bright field (video) microscopy during the process of carcinoma cell invasion of brain tissue. This model system also allows for cell exchange and replenishment approaches and offers a wide variety of manipulations and analyses.

Sistema Coculture con un cerebro rebanada organotípicos y 3D esferoide de células de carcinoma

Patients with cerebral metastasis of carcinomas have a poor prognosis. However, the process at the metastatic site has barely been investigated, in ...

Investigación

Medicina

High Throughput Characterization of Adult Stem Cells Engineered for Delivery of Therapeutic Factors for Neuroprotective Strategies

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to develop strategies for treating a variety of diseases. The purpose of this study was to develop and characterize MSCs as cellular vehicles engineered for delivery of therapeutic factors as part of a neuroprotective strategy for rescuing the damaged or diseased nervous system. In this study we used mouse MSCs that were genetically modified using lentiviral vectors, which encoded brain-derived neurotrophic factor (BDNF) or glial cell-derived neurotrophic factor (GDNF), together with green fluorescent protein (GFP). 
Before proceeding with in vivo transplant studies it was important to characterize the engineered cells to determine whether or not the genetic modification altered aspects of normal cell behavior. Different culture substrates were examined for their ability to support cell adhesion, proliferation, survival, and cell migration of the four subpopulations of engineered MSCs. High content screening (HCS) was conducted and image analysis performed. 
Substrates examined included: poly-L-lysine, fibronectin, collagen type I, laminin, entactin-collagen IV-laminin (ECL). Ki67 immunolabeling was used to investigate cell proliferation and Propidium Iodide staining was used to investigate cell viability. Time-lapse imaging was conducted using a transmitted light/environmental chamber system on the high content screening system.
Our results demonstrated that the different subpopulations of the genetically modified MSCs displayed similar behaviors that were in general comparable to that of the original, non-modified MSCs. The influence of different culture substrates on cell growth and cell migration was not dramatically different between groups comparing the different MSC subtypes, as well as culture substrates. 
This study provides an experimental strategy to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

This study describes an experimental platform to rapidly characterize engineered stem cells and their behaviors before their application in long-term in vivo transplant studies for nervous system rescue and repair.

Alto rendimiento caracterización de células madre adultas de ingeniería para la entrega de los factores terapéuticos de estrategias neuroprotectoras

Mesenchymal stem cells (MSCs) derived from bone marrow are a powerful cellular resource and have been used in numerous studies as potential candidates to ...

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 ...

A Brain Tumor/Organotypic Slice Co-culture System for Studying Tumor Microenvironment and Targeted Drug Therapies

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these tumors are not easily grown in standard culture conditions. Neurosphere cultures under serum-free conditions and orthotopic xenografts have expanded the range of tumors that can be maintained. However, many types of brain tumors remain difficult to propagate or study. This is particularly true for pediatric brain tumors such as pilocytic astrocytomas and medulloblastomas. This protocol describes a system that allows primary human brain tumors to be grown in culture. This quantitative assay can be used to investigate the effect of microenvironment on tumor growth, and to test new drug therapies. This protocol describes a system where fluorescently labeled brain tumor cells are grown on an organotypic brain slice from a juvenile mouse. The response of tumor cells to drug treatments can be studied in this assay, by analyzing changes in the number of cells on the slice over time. In addition, this system can address the nature of the microenvironment that normally fosters growth of brain tumors. This brain tumor organotypic slice co-culture assay provides a propitious system for testing new drugs on human tumor cells within a brain microenvironment.

Many types of human brain tumors are localized to specific regions within the brain and are difficult to grow in culture. This protocol addresses the role of tumor microenvironment and investigates new drug treatments by analyzing fluorescent primary brain tumor cells growing in an organotypic mouse brain slice. &#160;

Un tumor cerebral / organotípicos rebanada Co-cultura del sistema para estudiar microambiente tumoral y dirigida Medicamentos Terapias

Brain tumors are a major cause of cancer-related morbidity and mortality. Developing new therapeutics for these cancers is difficult, as many of these ...

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 ...

Optimized LC-MS/MS Method for the High-throughput Analysis of Clinical Samples of Ivacaftor, Its Major Metabolites, and Lumacaftor in Biological Fluids of Cystic Fibrosis Patients

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening pulmonary manifestations. Ivacaftor (IVA) and ivacaftor-lumacaftor (LUMA) combination are two new breakthrough CF drugs that directly modulate the activity and trafficking of the defective CFTR-protein. However, there is still a dearth of understanding on pharmacokinetic/pharmacodynamic parameters and the pharmacology of ivacaftor and lumacaftor. The HPLC-MS technique for the simultaneous analysis of the concentrations of ivacaftor, hydroxymethyl-ivacaftor, ivacaftor-carboxylate, and lumacaftor in biological fluids in patients receiving standard ivacaftor or ivacaftor-lumacaftor combination therapy has previously been developed by our group and partially validated to FDA standards. However, to allow the high-throughput analysis of a larger number of patient samples, our group has optimized the reported method through the use of a smaller pore size reverse-phase chromatography column (2.6 &#181;m, C8 100 &#197;; 50 x 2.1 mm) and a gradient solvent system (0-1 min: 40% B; 1-2 min: 40-70% B; 2-2.7 min: held at 70% B; 2.7-2.8 min: 70-90% B; 2.8-4.0 min: 90% B washing; 4.0-4.1 min: 90-40% B; 4.1-6.0 min: held at 40% B) instead of an isocratic elution. The goal of this study was to reduce the HPLC-MS analysis time per sample dramatically from ~15 min to only 6 min per sample, which is essential for the analysis of a large amount of patient samples. This expedient method will be of considerable utility for studies into the exposure-response relationships of these breakthrough CF drugs.

Ivacaftor and ivacaftor-lumacaftor combination are two new CF drugs. However, there is still a dearth of understanding on their PK/PD and pharmacology. We present an optimized HPLC-MS technique for the simultaneous analysis of ivacaftor and its major metabolites, and lumacaftor.

Método optimizado de LC-MS/MS para el análisis de alto rendimiento de las muestras clínicas de Ivacaftor, sus principales metabolitos y Lumacaftor en fluidos biológicos de pacientes con Fibrosis quística

Defects in the cystic fibrosis trans-membrane conductance regulator (CFTR) are the cause of cystic fibrosis (CF), a disease with life-threatening ...

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) models based on pBECs in mono-culture (MC), MC with astrocyte-conditioned medium (ACM), and non-contact co-culture (NCC) with astrocytes of porcine or rat origin. pBECs were isolated and cultured from fragments of capillaries from the brain cortices of domestic pigs 5-6 months old. These fragments were purified by careful removal of meninges, isolation and homogenization of grey matter, filtration, enzymatic digestion, and centrifugation. To further eliminate contaminating cells, the capillary fragments were cultured with puromycin-containing medium. When 60-95% confluent, pBECs growing from the capillary fragments were passaged to permeable membrane filter inserts and established in the models. To increase barrier tightness and BBB characteristic phenotype of pBECs, the cells were treated with the following differentiation factors: membrane permeant 8-CPT-cAMP (here abbreviated cAMP), hydrocortisone, and a phosphodiesterase inhibitor, RO-20-1724 (RO). The procedure was carried out over a period of 9-11 days, and when establishing the NCC model, the astrocytes were cultured 2-8 weeks in advance. Adherence to the described procedures in the protocol has allowed the establishment of endothelial layers with highly restricted paracellular permeability, with the NCC model showing an average transendothelial electrical resistance (TEER) of 1249 &#177; 80 &#937; cm2, and paracellular permeability (Papp) for Lucifer Yellow of 0.90 10-6 &#177; 0.13 10-6 cm sec-1 (mean &#177; SEM, n=55). Further evaluation of this pBEC phenotype showed good expression of the tight junctional proteins claudin 5, ZO-1, occludin and adherens junction protein p120 catenin. The model presented can be used for a range of studies of the BBB in health and disease and, with the highly restrictive paracellular permeability, this model is suitable for studies of transport and intracellular trafficking.

Improved Method for the Establishment of an In Vitro Blood-Brain Barrier Model Based on Porcine Brain Endothelial Cells

The aim of the protocol is to present an optimized procedure for the establishment of an in vitro blood-brain barrier (BBB) model based on primary porcine brain endothelial cells (pBECs). The model shows high reproducibility, high tightness, and is suitable for studies of transport and intracellular trafficking in drug discovery.

Mejor método para el establecimiento de un In Vitro Barrera Blood - Brain modelo basado en las células endoteliales de cerebro porcino

The aim of this protocol presents an optimized procedure for the purification and cultivation of pBECs and to establish in vitro blood-brain barrier (BBB) ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Aislamiento de células Decidual humanas primarias de las membranas fetales de la placenta de término

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

A High-Throughput Electrochemiluminescence 7-Plex Assay Simultaneously Screening for Type 1 Diabetes and Multiple Autoimmune Diseases

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate decarboxylase-65 (GADA), insulinoma antigen-2 (IA-2A), and zinc transporter-8 (ZnT8A) are equally important in disease prediction. Presently, up to 40% of patients diagnosed with T1D go on to develop other autoimmune disorders. Unfortunately, current screening methods using a single autoantibody for measurement are laborious and inefficient for large scale screening studies. We recently developed a simple multiplexed electrochemiluminescence (ECL) assay to address these current issues. The assay combines all 7 autoantibody tests into one well. Each well includes three IAbs (IAA, GADA, and IA-2A), autoantibodies to thyroid peroxidase (TPOA) and thyroid globulin (ThGA) to detect autoimmune thyroid disease, autoantibodies to tissue transglutaminase (TGA) for celiac disease, and autoantibodies to interferon alpha (IFN&#945;A) for autoimmune polyglandular syndrome-1 (APS-1); all of which screen for T1D and other relevant autoimmune diseases, simultaneously. The multiplex ECL assay is based on the single ECL assay platform, but instead uses the multiplex plate combining multiple autoantibody assays, up to 10, into a single well. The main difference from the single ECL assay is that each antibody-antigen complex formed in the fluid-phase is restrained onto a specific spot on each well through a linker system on the multiplex plate. The 7-Plex ECL assay, in the present study, is validated against standard radio-binding assays (RBA) and single ECL assays, using a large cohort of newly diagnosed T1D patients and age-matched healthy controls, resulting in excellent assay sensitivity and specificity.

We model a simple multiplexed ECL assay that combines 7 autoantibody assays together. The assay is capable of screening for T1D and multiple other autoimmune diseases, simultaneously, including celiac disease, autoimmune thyroid disease, and autoimmune polyglandular syndrome 1.

Un ensayo de electroquimioluminiscencia 7-plex de alto rendimiento que detecta simultáneamente la diabetes tipo 1 y múltiples enfermedades autoinmunes

Islet autoantibodies (IAbs) are widely used in type 1 diabetes (T1D) diagnosis and prediction. Four major IAbs to insulin (IAA), glutamate ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Caracterización de una nueva técnica de cultivo de retina organotípica humana

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...