Este trabajo fue apoyado por el Centro Nacional de Recursos para la Investigación y el Centro Nacional para el Avance de las Ciencias de traslación, los Institutos Nacionales de Salud, a través de subvención UL1TR000124 (FW); Felix y Mildred Yip Endowed Cátedra y el Fondo de la Familia Barnes (a DTWW), el Instituto Nacional de Odontología y de Investigación Craneofacial de los Institutos Nacionales de Salud, bajo el premio número T90DE022734 (MT). El contenido es responsabilidad exclusiva de los autores y no representa necesariamente las opiniones oficiales de los Institutos Nacionales de Salud.

1. magnética exosome extracción basado en perlas <ol><li> Pipetear una solución bien mezclada de 5 l de micropartículas magnéticas recubiertas con estreptavidina en 495 l de solución salina tamponada (PBS) tampón de fosfato en un tubo de microcentrífuga para resuspender las perlas. Lavar y resuspender las perlas con 500 l de PBS tres veces usando una rejilla magnética. El bastidor es un conjunto de imanes en el lado de una unidad de vivienda que puede sostener los tubos de muestra de microcentrífuga. <ol><li> Para cada lavado, primero deje que los tubos se sientan en el estante durante 1 minuto, y luego usar una punta de pipeta para retirar con cuidado el tampón sobrenadante sin perturbar las cuentas. </li><li> Colocar los tubos en un estante regular sin imanes en el lado. Añadir 500 l de PBS en los tubos, y el uso de la pipeta para mezclar la solución y los granos juntos. A continuación, poner los tubos de nuevo en la gradilla magnética para separar de nuevo las perlas de la solución. </li><li> Realizar esta eliminación de tampón a través de la magnetización y resuspensión en PBS unatotal de tres veces. Esto realiza un lavado inicial de las partículas magnéticas. </li></ol></li><li> Resuspender las perlas en 490 l de tampón de PBS, con el tubo colocado en la parte no magnetizada de la gradilla magnética. Pipetear 5 l de ratón biotinilado anticuerpo anti-CD63 humano en 1,0 mg / ml de concentración de stock en la mezcla de perlas. Use la pipeta para mezclar las perlas y el anticuerpo en solución. </li><li> Coloque los tubos de microcentrífuga con el grano y la mezcla de anticuerpos con biotina en un rotador de muestras. Establezca los parámetros de los rotadores para el rotador de la muestra para la rotación recíproca a 90 ° de inclinación durante 5 segundos y vibrando a 5 ° durante 1 seg. Rotar los tubos de mezcla de muestra de perlas en estos parámetros para 30 min a TA. </li><li> Eliminar el anticuerpo no unido después de la conjugación. <ol><li> Después de 30 min de rotación a TA, coloque los tubos de nuevo en la gradilla magnética durante 5 min. </li><li> Realizar tres lavados de los granos mediante la eliminación de la fase líquida utilizando una micropipeta y se lava con 500 μ; L de PBS. Después de la triple lavado, volver a suspender las perlas en 490 l de caseína-PBS y colocar en la parte no magnetizado de la cremallera. </li></ol></li><li> Extracción exosome usando perlas recubiertas de anticuerpo. <ol><li> Marque cada tubo con específica identificación de la muestra. Pipetear una muestra de 10 l de suero o saliva en el tubo de microcentrífuga. Use la pipeta para mezclar la muestra y perlas magnéticas pipeteando varias veces. </li><li> Colocar los tubos con la muestra y anti-humanos perlas de anticuerpo CD63 en los rotadores y rotar durante 2 horas a RT. Utilice los mismos parámetros de los rotadores, como se describe en el paso 1.2. </li><li> Después de 2 hr de la muestra de rotación, realizar un lavado triple de magnetización a perlas separadas de la solución, la eliminación de fase líquida con micropipeta, y resuspensión de perlas en 500 l de tampón Tris-HCl. Las perlas resultantes están ligados a los exosomas y están listos para la liberación del campo eléctrico y la medición. </li></ol></li></ol> 2. Electric Field inducida publicó unnd Medición de exosomal contenido <ol><li> Inicial PRECOATING de Electrodo con GADPH Primer <ol><li> Aplicar un plástico bien a un conjunto de electrodos para evitar la contaminación cruzada de los electrodos individuales. Para este experimento, utilizar un conjunto de electrodos 16-sensor con cada electrodo unidad en la matriz que consiste en un trabajo, contador, y electrodo de referencia de oro desnudo. </li><li> Preparar una mezcla madre de 100 Nm sonda de ADN, 0,3 M KCl, y pirrol 10 mM pipeteando reactivos de valores en un tubo con agua destilada ultrapura. Mezclar bien por agitación. NOTA: Para este estudio, la sonda de ADN seleccionada corresponde a la referencia de genes GAPDH, que se sabe que existe dentro de los exosomas. La secuencia de la sonda utilizada es: 5&#39;-Biotina-AGGTCCACCACTGACACGTTG-3 &#39;. Utilice esta mezcla sobre todos los electrodos. </li><li> Pipetear 60 l de la mezcla de sondas de ADN-monómero sobre la superficie de cada electrodo de oro. Examinar los electrodos para garantizar que hay una cobertura adecuada de la de trabajo, counter, y de referencia de la mezcla líquida. </li><li> Electropolymerize mezcla de monómeros-sonda para crear una capa de polímero conductor en la superficie del electrodo mediante la aplicación de una onda cuadrada (CSW) el perfil de campo eléctrico cíclico a la superficie del electrodo. Este campo eléctrico consiste en aplicar 350 mV durante 9 s e inmediatamente cambiar a 950 mV durante 1 seg. Aplicar este perfil cíclico de onda cuadrada al electrodo durante 10 ciclos, para un total de 100 seg de campo eléctrico aplicado. </li><li> Enjuague superficie del sensor 3 veces con agua destilada y se seca con gas nitrógeno para eliminar el líquido de la superficie del electrodo. Asegúrese de que el líquido se elimina correctamente del electrodo. </li></ol></li><li> Descarga exosome Cargo <ol><li> Cargar 5 l de 1 M de una sonda detector en 495 l de la mezcla de complejo de grano exosome y utilizar una pipeta para mezclar. NOTA: La sonda detector es una secuencia de cebador de ADN conjugado con una molécula de fluoresceína en el extremo 3 &#39;. El prob detectorsecuencia e utilizado para este estudio corresponde al ARNm GAPDH se encuentra dentro de los exosomas. La secuencia de la sonda detector conjugado con fluoresceína es: 5&#39;-GCAGTGGGGACACGGAAGGCC-fluoresceína-3 &#39;. </li><li> Pipeta 60 l de la sonda y compleja mezcla grano-exosome sobre una superficie de electrodos de oro con una selección de imanes debajo. Esta matriz imán consta de dieciséis 2,54 mm imanes de neodimio diámetro alineadas para corresponder a los electrodos de trabajo del sensor. La Figura 2A ilustra la colocación de los imanes y solución de grano exosome. </li><li> Una vez que la muestra se carga en la superficie del electrodo, se aplican 20 ciclos del campo eléctrico CSW con 9 segundos a -300 mV y 1 seg a 200 mV (200 sec total). La carga exosomal que se libera se hibridará con los cebadores en la superficie del electrodo. Si los marcadores de superficie de la exosome son objeto de investigación, omitir esta parte del experimento. La Figura 2B ilustra este proceso. </li> &lt;li&gt; Wash-off los analitos no unidos en la superficie del electrodo con un triple enjuague la superficie del electrodo con agua destilada. Secar el electrodo con gas nitrógeno. </li></ol></li><li> Reportero de anticuerpos y de lectura <ol><li> Añadir 60 l de 150 unidades / anticuerpo anti-fluoresceína ml conjugado con peroxidasa de rábano (HRP en 1: 1000 dilución) diluidas en caseína / PBS. </li><li> Utilice una conjugación impulsado campo eléctrico a lo complejo HRP anti-fluoresceína para el bocadillo de la sonda. Aplicar -200 mV durante 1 segundo y 500 mV durante 1 segundo durante 5 ciclos a la superficie del electrodo. La Figura 2A muestra los complejos de sonda de captura y detector para tanto una proteína y sistema de ácido nucleico. </li><li> Triple superficie del sensor de lavado usando agua destilada y se seca con gas nitrógeno. </li><li> Después de la eliminación por lavado el exceso de anticuerpo no unido de anti-fluoresceína, añadir 60 l de 3,3 &#39;, 5,5&#39;-tetrametilbenzidina (TMB). Cargar este sustrato a cada superficie del sensor usando una pipeta multicanal. </li><li> Realizar la lectura amperométrica de la corriente mediante la medición de la corriente del electrodo en -200 mV durante 60 segundos usando un potenciostato electroquímico capaz de la medición simultánea de 16 canales. Figura 2C es un ejemplo de perfil actual durante la lectura. </li></ol></li></ol><img alt="Figura 2" src="/files/ftp_upload/52439/52439fig2highres.jpg" /> Figura 2. Componentes del Método Efirm. (A) método de extracción de exosomas de biofluido usando micropartículas magnéticas CD63 anti-humano recubierto y luego la descarga de la carga exosome usando ondas cuadradas cíclicos aplicados al complejo de partícula exosome. (B) Esquema de biosensor electrodo utilizado para la detección de objetivos de ARN / ADN / proteína de la exosome liberado. (C) Ejemplo representativo de lectura amperométrica de la metodología Efirm, donde la magnitud de corriente más grande corresponde to niveles más altos de una biomolécula. Esta cifra es de Wei et al. 14 <a href="https://www.jove.com/files/ftp_upload/52439/52439fig2large.jpg" target="_blank">Por favor haga clic aquí para ver una versión más grande de esta figura.</a>

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breast+Cancer+Exosome-like+Microvesicles+and+Salivary+Gland+Cells+Interplay+Alters+Salivary+Gland+Cell-Derived+Exosome-like+Microvesicles+In.">Breast Cancer Exosome-like Microvesicles and Salivary Gland Cells Interplay Alters Salivary Gland Cell-Derived Exosome-like Microvesicles In.</a> Vitro. PLoS ONE. 7 (3), e33037 (2012).</li><li>Bala, S., 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=Circulating+microRNAs+in+exosomes+indicate+hepatocyte+injury+and+inflammation+in+alcoholic,+drug-induced,+and+inflammatory+liver+diseases.">Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic, drug-induced, and inflammatory liver diseases.</a> Hepatolog. 56 (5), 1946-1957 (2012).</li><li>Dear, J. W., Street, J. 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David Wong es co-fundador de RNAmeTRIX Inc., una compañía de diagnóstico molecular. PeriRx LLC sublicenciada propiedad intelectual relacionados con el diagnóstico molecular de RNAmeTRIX. David Wong es un consultor para PeriRx.

Como indican los resultados, las nanopartículas magnéticas CD63 anti-humano recubierto son capaces de capturar específicamente pequeñas partículas que tienen un tamaño que varía desde 70 hasta 100 nm. Esta partícula capturado es consistente con el perfil observado previamente de exosomas. Además, se muestra el uso de la CSW de baja tensión después de la captura de las partículas para eliminarlos de la superficie de la perla y causar perfiles de degradación de ADN similares a la de un método tradicional bas...

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>. The disclosures were updated.

An erratum was issued for <a href="http://www.jove.com/video/52439">Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)</a>.

Errata: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

Investigación exosome es un campo emergente de investigación que examina microvesículas lípidos que llevan ARN 1, DNA 2, y la proteína 3 de carga. Investigaciones anteriores de la biología exosome han conducido a la identificación de los exosomas en biofluidos tales como sangre 4, 5 orina, leche materna 6, 7 y la saliva. Los estudios han demostrado que los exosomas juegan un papel en diferentes vías celulares, meditando remotamente la comunicación entre diferentes sistemas del cuerpo 8. Debido al papel exosomas desempeñan en la comunicación intercelular, se plantea la hipótesis de que pueden empaquetar objetivos de biomoléculas (proteínas, ARN y ADN) correlacionados con estados de enfermedad. In vitro 3 y animal modelo de 9 estudios parecen corroborar esta hipótesis. En la investigación de contenido exosomal para el descubrimiento de biomarcadores, es necesario el desarrollo de una metodología para el aislamiento selectivo de exosome biofluidos, expulsi inducidaen la carga de los exosomas, y cuantificación de biomoléculas de exosoma. En la medida de este trabajo, los exosomas se definirán como una estructura que tiene un diámetro de aproximadamente 70-100 nm y poseyendo la superficie marcador CD63. Los investigadores típicamente primera purifican exosomas por ultracentrifugación 10 y luego procesar el contenido exosomal través de la utilización de kits de tampón de lisis. El uso de métodos de tampón de lisis requiere tiempos de incubación que van desde minutos a horas. Este proceso puede potencialmente dañar carga exosome y conducir a probar la degradación. Por ejemplo, el ARN exosome salival lanzado a través de tampón de lisis en el entorno extracelular que rodea posee una vida media de menos de 1 min, por lo que la medición de RNA exosomal post-Tampón de Lisis una tarea particularmente difícil sin la adición de reactivos de estabilización 11. El efecto combinado de la adición de diversos reactivos para la lisis y la estabilización puede introducir agentes que complican e interferir con la Analysis de contenido exosomal. Un enfoque alternativo puede ser útil para la descarga rápidamente contenido exosomal y conservar de forma segura la carga para su caracterización. En este trabajo, se propone el uso de un campo eléctrico no uniforme para la liberación de contenido exosomal. Eléctricos campos han sido conocidos por llevar la capacidad de polarizar y perturbar la bicapa lipídica que forma las membranas celulares. Nuestro trabajo experimental explora el uso de ondas cuadradas cíclicos no uniformes (CSW) para interrumpir la estructura microvesícula de exosomas y la liberación de la carga transportada. Este método utiliza tensiones en el rango de varios cientos de milivoltios, lo que significa que la mayoría de las biomoléculas no serán interrumpidos. Se demuestra que el uso de una onda cíclica cuadrados es capaz de accionar la liberación de exosome salival contenido de mRNA en el medio ambiente fluido circundante. Esta versión de contenido exosomal se integra perfectamente con un sistema de electrodos que puede ser utilizado para cuantificar los niveles de expresión de biomarcador 12,13. Este método propuesto permite una rápida sensible, y el tampón de lisis análisis, libres de contenidos exosome. <img alt="Figura 1" src="/files/ftp_upload/52439/52439fig1highres.jpg" /> Figura 1. Visión general de Efirm de flujo de trabajo.. El método Efirm se divide a grandes rasgos en las tres fases principales que son necesarios para la purificación y el análisis de los exosomas. Este método de liberación contenido exosomal y el análisis basado CSW se utiliza en conjunción con microperlas magnéticas-CD63 específico para el aislamiento exosome. Estas perlas CD63-de afinidad permiten el aislamiento selectivo de los exosomas de las muestras salivales (y otros fluidos biológicos). Después de la incubación y la extracción de los exosomas utilizando las perlas magnéticas, las cuentas se migran al sistema de sensor electroquímico para la CSW basado comunicado de contenido y parte de análisis del experimento. Figura 1 ofrece una visión general de la obrade flujo del método Efirm.

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		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Name of Material/ Equipment</td>
			<td style="width:157px;">Company</td>
			<td style="width:132px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Helios 16-Channel Reader System with Chip Interface&#160;</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td style="width:132px;">&#160;RS-1000-16</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">16X Sensor Chip, Bare Gold, pack of 5 chips</td>
			<td style="width:157px;">Genefluidics, USA</td>
			<td>SC1000-16X-B</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Biotinylated anti-human CD63 Antibody</td>
			<td style="width:157px;">Ancell, USA</td>
			<td style="width:132px;">215-030</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Dynabeads MyOne Streptavidin T1</td>
			<td>Invitrogen, USA</td>
			<td>65601</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Neodynium Magnetics ( 1/10&#34; dia. x 1/32&#34; thick)</td>
			<td style="width:157px;">K&#38;J Magnetics, USA</td>
			<td>DH101</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Ultrapure Distilled Water</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td>10977-023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Mettler Toldeo 3M KcL Solution</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>1911512</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Pyrrole</td>
			<td style="width:157px;">Sigma-Aldrich, USA</td>
			<td style="width:132px;">W338605-100g</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Anti-Fluorescein-POD, Fab fragments</td>
			<td style="width:157px;">Roche, Germany</td>
			<td>11426346910</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:347px;">3, 3&#8242;, 5, 5&#8242; tetramethylbenzidine substrate (TMB/H2O2, low activity)&#160;</td>
			<td style="width:157px;">Neogen, Usa</td>
			<td style="width:132px;">330175</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:347px;">Phosphate Buffered Saline Solution</td>
			<td style="width:157px;">Life Technologies, USA</td>
			<td align="right" style="width:132px;">10010023</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:347px;">Casein/PBS</td>
			<td style="width:157px;">Fisher Scientific, USA</td>
			<td>37532</td>
			<td></td>
		</tr>
	</tbody>
</table>

detection of exosomal biomarker by electric field-induced release and measurement (efirm)

Los exosomas son estructuras microvesicular que juegan un papel mediador en la comunicación intercelular. Es de interés para el estudio de la carga interna de exosomas para determinar si llevan biomarcadores discriminatorias de enfermedades. Para la realización de análisis exosomal, es necesario desarrollar un método para la extracción y el análisis de los exosomas de biofluidos objetivo sin dañar el contenido interno. Liberación inducida por el campo eléctrico y medición (Efirm) es un método para extraer específicamente exosomas de biofluidos, descargando su carga, y prueba de su contenido interno de ARN / proteína. El uso de un anticuerpo micropartícula magnética específica anti-CD63 humano, los exosomas son primero precipitan a partir de biofluidos. Después de la extracción, las ondas de baja tensión eléctricos cíclicos cuadrados (CSW) se aplican a romper la membrana vesicular y causar desembarque de la carga. El contenido de la exosome se hibrida con cebadores de ADN o anticuerpos inmovilizados sobre una superficie de electrodo para quantification del contenido molecular. El método Efirm es ventajoso para la extracción de los exosomas y descargando una carga para el análisis sin tampón de lisis. Este método es capaz de realizar la detección específica de ambas dianas de ARN y proteína de biomarcadores en el exosome. Efirm extrae exosomas específicamente en base a sus marcadores de superficie en comparación con las técnicas de tamaño basada en acciones. Microscopía electrónica de transmisión (TEM) y el ensayo demuestran la funcionalidad del método para la captura y análisis exosome. El método se aplicó a Efirm exosomal análisis de 9 ratones inyectados con células de pulmón humano H640 cáncer (una línea celular transfectada para expresar el marcador exosome humano CD63-GFP) con el fin de poner a prueba su perfil exosome contra 11 ratones que recibieron los controles salinos. Se hallaron niveles elevados de biomarcadores exosomal (referencia GAPDH gen y la proteína marcador de superficie CD63 humano-GFP) para el H640 ratones inyectados en ambas muestras de suero y saliva. Además, saliva y muestras de suero se demostró tener linealidad (R = 0,79). Estos resultados son indicativos para la viabilidad de los biomarcadores de exosoma salivales para la detección de enfermedades distales.

Validación de exosome captura de perlas usando TEM El aislamiento de los exosomas de saliva usando perlas magnéticas CD63 anti-humano fue validado siguiendo el protocolo de extracción mediante el uso de microscopía electrónica de transmisión (TEM). TEM muestra perlas magnéticas con 70-100 nm gránulos inmediatamente adyacentes (ver Figura 3A, y 3B), en consonancia con el perfil conocido de exosomas. No se observaron gránulos 70-100 n...

Watch this Scientific Journal Video about Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM at JoVE.com

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves.

La detección de biomarcadores exosomal por inducida por campo eléctrico de lanzamiento y Medición (Efirm)

Exosomes are microvesicular structures that play a mediating role in intercellular communication. It is of interest to study the internal cargo of ...

detection-exosomal-biomarker-electric-field-induced-release

Research

JoVE Journal

Bioengineering

Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement (EFIRM)

18.3K vistas.  University of California, Los Angeles. Exosomes are microvesicular structures found within biofluids that potentially carry important disease discriminatory biomarkers. Here, a novel method is used to specifically extract exosomes and rapidly test the exosomal cargo for both RNA/protein targets following the disruption of exosomes using non-uniform electric cyclic square waves. 

Video: Detection of Exosomal Biomarker by Electric Field-induced Release and Measurement EFIRM

Developing Custom Chinese Hamster Ovary-host Cell Protein Assays using Acoustic Membrane Microparticle Technology

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to the complexity of development on alternate platforms. BioScale's proprietary Acoustic Membrane MicroParticle (AMMP) technology allows sandwich immunoassays to be easily developed for use on the ViBE platform, providing better sensitivity, reproducibility, and automated operation. Provided as an example, this protocol outlines the procedure for developing a custom Chinese Hamster Ovary- Host Cell Protein (CHO-HCP) assay. The general principles outlined here can be followed for the development of a wide variety of immunoassays.
An AMMP assay measures antigen concentration by measuring changes in oscillation frequency caused by the binding of microparticles to the sensor surface to calculate. It consists of four major components: (1) a cartridge that contains a functionalized eight sensor chip (2) antibody labeled magnetic microparticles, (3) hapten tagged antibody that binds to the surface of the functionalized chip (4) samples containing the antigen of interest. BioScale's biosensor is a resonant device that contains eight individual membranes with separate fluidic paths. The membranes change oscillation frequency in response to mass accumulating on the surface and this frequency change is used to quantitate the amount of added mass.
To facilitate use in a wide variety of immunoassays the sensor is functionalized with an anti-hapten antibody. Assay specific antibodies are modified through the covalent conjugation of a hapten tag to one antibody and biotin to the other. The biotin label is used to bind the antibody to streptavidin coupled magnetic beads which, in combination with the hapten-tagged antibody, are used to capture the analyte in a sandwich. The complex binds to the chip through the anti-hapten/hapten interaction. At the end of each assay run the sensors are cleaned with a dilute acid enabling the sequential analysis of columns from a 96-well plate.
Here, we present the method for developing a custom CHO-HCP AMMP assay for bioprocess development. Developing AMMP assays or modifying existing assays into AMMP assays can provide better performance (reproducibility, sensitivity) in complex samples and reduced operator time. The protocol shows the steps for development and the discussion section reviews representative results. For a more in-depth explanation of assay optimization and customization parameters contact BioScale. This kit offers generic bioprocess development assays such as Residual Protein A, Product titer, and CHO-HCP.

Development of custom assays on the ViBE platform for more sensitive, reproducible, automated results in complex matrices is described. The universal cartridge allows assays to be easily adapted for use with custom assays. This versatility enables rapid development and validation of novel assays or automated versions of existing manual assays, exemplified in this video.

Desarrollar ovario de hámster chino anfitrión-ensayos con células de proteínas utilizando la tecnología de membrana acústica de micropartículas

Custom assays for unique proteins are often limited to time consuming manual detection and quantitation techniques such as ELISA or Western blots due to ...

Investigación

Bioingeniería

Measurement of Aggregate Cohesion by Tissue Surface Tensiometry

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium. We have developed tissue surface tensiometry (TST) specifically to measure the surface free energy of interaction between cells. The biophysical concepts underlying TST have been previously described in detail1,2. The method is based on the observation that mutually cohesive cells, if maintained in shaking culture, will spontaneously assemble into clusters. Over time, these clusters will round up to form spheres. This rounding-up behavior mimics the behavior characteristic of liquid systems. Intercellular binding energy is measured by compressing spherical aggregates between parallel plates in a custom-designed tissue surface tensiometer. The same mathematical equation used to measure the surface tension of a liquid droplet is used to measure surface tension of 3D tissue-like spherical aggregates. The cellular equivalent of liquid surface tension is intercellular binding energy, or more generally, tissue cohesivity. Previous studies from our laboratory have shown that tissue surface tension (1) predicts how two groups of embryonic cells will interact with one another1-5, (2) can strongly influence the ability of tissues to interact with biomaterials6, (3) can be altered not only through direct manipulation of cadherin-based intercellular cohesion7, but also by manipulation of key ECM molecules such as FN8-11 and 4) correlates with invasive potential of lung cancer12, fibrosarcoma13, brain tumor14 and prostate tumor cell lines15. In this article we will describe the apparatus, detail the steps required to generate spheroids, to load the spheroids into the tensiometer chamber, to initiate aggregate compression, and to analyze and validate the tissue surface tension measurements generated.

We describe a method of measuring binding energy, expressible as tissue surface tension, between cells within 3D tissue-like aggregates. Differences in tissue surface tension have been demonstrated to correlate with invasiveness of lung, muscle, and brain tumors, and are fundamental determinants of establishing spatial relationships between different cell types.

La medición de la cohesión agregado por tensiometría tejido de la superficie

Rigorous measurement of intercellular binding energy can only be made using methods grounded in thermodynamic principles in systems at equilibrium.  We ...

Measurement of Tension Release During Laser Induced Axon Lesion to Evaluate Axonal Adhesion to the Substrate at Piconewton and Millisecond Resolution

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is subject to chemical and mechanical signals, and the mechanisms by which it senses and responds to mechanical signals are poorly understood. Elucidating the role of forces in cell maturation will enable the design of scaffolds that can promote cell adhesion and cytoskeletal coupling to the substrate, and therefore improve the capacity of different neuronal types to regenerate after injury.
Here, we describe a method to apply simultaneous force spectroscopy measurements during laser induced cell lesion. We measure tension release in the partially lesioned axon by simultaneous interferometric tracking of an optically trapped probe adhered to the membrane of the axon. Our experimental protocol detects the tension release with piconewton sensitivity, and the dynamic of the tension release at millisecond time resolution. Therefore, it offers a high-resolution method to study how the mechanical coupling between cells and substrates can be modulated by pharmacological treatment and/or by distinct mechanical properties of the substrate.

We measured the tension release in an axon that was partially lesioned with a laser dissector by simultaneous force spectroscopy measurement performed on an optically-trapped probe adhered to the membrane of the axon. The developed experimental protocol evaluates the axon adhesion to the culture substrate.

Medición de la tensión de salida durante Laser Induced Axon lesión axonal a evaluar la adhesión al sustrato en picoNewton y Millisecond Resolución

The formation of functional connections in a developing neuronal network is influenced by extrinsic cues. The neurite growth of developing neurons is ...

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a model microorganism. The biosensor relies on direct binding of the target bacteria cells onto its surface, while no pretreatment (e.g.&#160;by cell lysis) of the studied sample is required. A mesoporous Si thin film is used as the optical transducer element of the biosensor. Under white light illumination, the porous layer displays well-resolved Fabry-P&#233;rot fringe patterns in its reflectivity spectrum. Applying a fast Fourier transform (FFT) to reflectivity data results in a single peak. Changes in the intensity of the FFT peak are monitored. Thus, target bacteria capture onto the biosensor surface, through antibody-antigen interactions, induces measurable changes in the intensity of the FFT peaks, allowing for a &#39;real time&#39; observation of bacteria attachment.
The mesoporous Si film, fabricated by an electrochemical anodization process, is conjugated with monoclonal antibodies, specific to the target bacteria. The immobilization, immunoactivity and specificity of the antibodies are confirmed by fluorescent labeling experiments. Once the biosensor is exposed to the target bacteria, the cells are directly captured onto the antibody-modified porous Si surface. These specific capturing events result in intensity changes in the thin-film optical interference spectrum of the biosensor. We demonstrate that these biosensors can detect relatively low bacteria concentrations (detection limit of 104 cells/ml) in less than an hour.

Optical Detection of E. coli Bacteria by Mesoporous Silicon Biosensors

A label-free optical biosensor for rapid bacteria detection is introduced. The biosensor is based on a nanostructured porous Si, which is designed to directly capture the target bacteria cells onto its surface. We use monoclonal antibodies, immobilized onto the porous transducer, as the capture probes. Our studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations within minutes with no prior sample processing (such as cell lysis).

Detección óptica de<em&gt; E. coli</em&gt; Bacterias por mesoporosos Silicon Biosensores

A label-free optical biosensor based on a nanostructured porous Si is designed for rapid capture and detection of Escherichia coli K12 bacteria, as a ...

Measurement of Maximum Isometric Force Generated by Permeabilized Skeletal Muscle Fibers

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. Single muscle fiber studies are useful in both basic science and clinical studies. For basic studies, single muscle fiber contractility measurements allow investigation of fundamental mechanisms of force production, and analysis of muscle function in the context of genetic manipulations. Clinically, single muscle fiber studies provide useful insight into the impact of injury and disease on muscle function, and may be used to guide the understanding of muscular pathologies. In this video article we outline the steps required to prepare and isolate an individual skeletal muscle fiber segment, attach it to force-measuring apparatus, activate it to produce maximum isometric force, and estimate its cross-sectional area for the purpose of normalizing the force produced.

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle function at the level of the single muscle cell. In this article we outline a valid and reliable technique to prepare and test permeabilized skeletal muscle fibers in vitro.

Medición de la fuerza isométrica máxima generada por las fibras musculares esqueléticas permeabilizadas

Analysis of the contractile properties of chemically skinned, or permeabilized, skeletal muscle fibers offers a powerful means by which to assess muscle ...

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate (LAL) Assays

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause inflammation, fever, hypo- or hypertension, and, in extreme cases, can lead to tissue and organ damage that may become fatal. The amounts of endotoxin in pharmaceutical products, therefore, are strictly regulated. Among the methods available for endotoxin detection and quantification, the Limulus Amoebocyte Lysate (LAL) assay is commonly used worldwide. While any pharmaceutical product can interfere with the LAL assay, nano-formulations represent a particular challenge due to their complexity. The purpose of this paper is to provide a practical guide to researchers inexperienced in estimating endotoxins in engineered nanomaterials and nanoparticle-formulated drugs. Herein, practical recommendations for performing three LAL formats including turbidity, chromogenic and gel-clot assays are discussed. These assays can be used to determine endotoxin contamination in nanotechnology-based drug products, vaccines, and adjuvants.

Detection of Endotoxin in Nano-formulations Using Limulus Amoebocyte Lysate LAL Assays

Detection of endotoxins in engineered nanomaterials represents one of the grand challenges in the field of nanomedicine. Here, we present a case study that describes the framework composed of three different LAL formats to estimate potential endotoxin contamination in nanoparticles.

Detección de endotoxinas en las formulaciones Nano mediante ensayos de Limulus Amoebocyte Lysate (LAL)

When present in pharmaceutical products, a Gram-negative bacterial cell wall component endotoxin (often also called lipopolysaccharide) can cause ...

Electric-Field-Induced Neural Precursor Cell Differentiation in Microfluidic Devices

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell culture system that was used for a long-term cell differentiation study using microscopy. The microfluidic system consists of the following major components: an optically transparent electrotactic chip, a transparent indium-tin-oxide (ITO) heater, a culture media-filling pump, an electrical power supply, a high-frequency power amplifier, an EF multiplexer, a programmable X-Y-Z motorized stage, and an inverted phase-contrast microscope equipped with a digital camera. The microfluidic system is beneficial in simplifying the overall experimental setup and, in turn, the reagent and sample consumption. This work involves the differentiation of neural stem and progenitor cells (NPCs) induced by direct current (DC) pulse stimulation. In the stem cell maintenance medium, the mouse NPCs (mNPCs) differentiated into neurons, astrocytes, and oligodendrocytes after the DC pulse stimulation. The results suggest that simple DC pulse treatment could control the fate of mNPCs and could be used to develop therapeutic strategies for nervous system disorders. The system can be used for cell culture in multiple channels, for long-term EF stimulation, for cell morphological observation, and for automatic time-lapse image acquisition. This microfluidic system not only shortens the required experimental time, but also increases the accuracy of control on the microenvironment.

In this study, we present a protocol for the differentiation of neural stem and progenitor cells (NPCs) solely induced by direct current (DC) pulse stimulation in a microfluidic system.

Diferenciación celular precursora neuronal inducida por campo eléctrico en dispositivos microfluídicos

Physiological electric fields (EF) play vital roles in cell migration, differentiation, division, and death. This paper describes a microfluidic cell ...

Finite Element Modelling of a Cellular Electric Microenvironment

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the mechanisms of cell response when exposed to electrical fields can therefore guide the optimization of clinical applications. In vitro experiments aim to help uncover those, offering the advantage of wider input and output ranges that can be ethically and effectively assessed. However, the advancements in in vitro experiments are difficult to reproduce directly in clinical settings. Mainly, that is because the ES devices used in vitro differ significantly from the ones suitable for patient use, and the path from the electrodes to the targeted cells is different. Translating the in vitro results into in vivo procedures is therefore not straightforward. We emphasize that the cellular microenvironment's structure and physical properties play a determining role in the actual experimental testing conditions and suggest that measures of charge distribution can be used to bridge the gap between in vitro and in vivo. Considering this, we show how in silico finite element modelling (FEM) can be used to describe the cellular microenvironment and the changes generated by electric field (EF) exposure. We highlight how the EF couples with geometric structure to determine charge distribution. We then show the impact of time dependent inputs on charge movement. Finally, we demonstrate the relevance of our new in silico model methodology using two case studies: (i) in vitro fibrous Poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT-PSS) scaffolds and (ii) in vivo collagen in extracellular matrix (ECM).

This paper presents a strategy for building finite element models of fibrous conductive materials exposed to an electric field (EF). The models can be used to estimate the electrical input that cells seeded in such materials receive and assess the impact of changing the scaffold's constituent material properties, structure or orientation.

Modelado de elementos finitos de un microambiente eléctrico celular

Clinical studies show electrical stimulation (ES) to be a potential therapy for the healing and regeneration of various tissues. Understanding the ...

Electric and Magnetic Field Devices for Stimulation of Biological Tissues

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, differentiation, morphology, and molecular synthesis. However, variables such stimuli strength and stimulation times need to be considered when stimulating either cells, tissues or scaffolds. Given that EFs and MFs vary according to cellular response, it remains unclear how to build devices that generate adequate biophysical stimuli to stimulate biological samples. In fact, there is a lack of evidence regarding the calculation and distribution when biophysical stimuli are applied. This protocol is focused on the design and manufacture of devices to generate EFs and MFs and implementation of a computational methodology to predict biophysical stimuli distribution inside and outside of biological samples. The EF device was composed of two parallel stainless-steel electrodes located at the top and bottom of biological cultures. Electrodes were connected to an oscillator to generate voltages (50, 100, 150 and 200 Vp-p) at 60 kHz. The MF device was composed of a coil, which was energized with a transformer to generate a current (1 A) and voltage (6 V) at 60 Hz. A polymethyl methacrylate support was built to locate the biological cultures in the middle of the coil. The computational simulation elucidated the homogeneous distribution of EFs and MFs inside and outside of biological tissues. This computational model is a promising tool that can modify parameters such as voltages, frequencies, tissue morphologies, well plate types, electrodes and coil size to estimate the EFs and MFs to achieve a cellular response.

This protocol describes the step-by-step process to build both electrical and magnetic stimulators used to stimulate biological tissues. The protocol includes a guideline to simulate computationally electric and magnetic fields and manufacture of stimulator devices.

Dispositivos eléctricos y de campo magnético para la estimulación de tejidos biológicos

Electric fields (EFs) and magnetic fields (MFs) have been widely used by tissue engineering to improve cell dynamics such as proliferation, migration, ...

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

The ability to measure biomarkers in vivo relevant to the assessment of disease progression is of great interest to the scientific and medical communities. The resolution of results obtained from current methods of measuring certain biomarkers can take several days or weeks to obtain, as they can be limited in resolution both spatially and temporally (e.g., fluid compartment microdialysis of interstitial fluid analyzed by enzyme-linked immunosorbent assay [ELISA], high-performance liquid chromatography [HPLC], or mass spectrometry); thus, their guidance of timely diagnosis and treatment is disrupted. In the present study, a unique technique for detecting and measuring peptide transmitters in vivo through the use of a capacitive immunoprobe biosensor (CI probe) is reported. The fabrication protocol and in vitro characterization of these probes are described. Measurements of sympathetic stimulation-evoked neuropeptide Y (NPY) release in vivo are provided. NPY release is correlated to the sympathetic release of norepinephrine for reference. The data demonstrate an approach for the fast and localized measurement of neuropeptides in vivo. Future applications include intraoperative real-time assessment of disease progression and minimally invasive catheter-based deployment of these probes.

Time-Resolved In Vivo Measurement of Neuropeptide Dynamics by Capacitive Immunoprobe in Porcine Heart

Established immunochemical methods to measure peptide transmitters in vivo rely on microdialysis or bulk fluid draw to obtain the sample for offline analysis. However, these suffer from spatiotemporal limitations. The present protocol describes the fabrication and application of a capacitive immunoprobe biosensor that overcomes the limitations of the existing techniques.