1. modificación polimérico Superficies con recCD47 NOTA:. El protocolo se resume esquemáticamente en la Figura 1 La Figura 1A ilustra la generación de superficies poliméricos reactivos con tiol Figura 1B ilustra la generación de recCD47 reactivo con tiol.. <ol><li> Día 1 <ol><li> Preparar una solución de PDT-BzPh (1 mg / ml) y bicarbonato de potasio (KHCO3) (0,7 mg / ml) en agua estéril. Revuelva la noche a 4 ° C (protegerlo de la luz). </li></ol></li><li> Día 2 <ol><li> Cortar tubo polimérico en 40 cm piezas largas (suficiente largo para caber alrededor de las ruedas giratorias). </li><li> Remojar tubos en una solución acuosa al 0,1% de hexacylpyridinium durante 90 min a temperatura ambiente en un agitador o en el aparato de Chandler Loop. Enjuague los tubos con 3x de agua estéril después de la 90 min remojo. </li><li> Se acidifica la solución de PDT-BzPh desde el día 1 por la adición de fosfato monobásico de potasio acuoso al 15% (KH 2 </sub&gt; PO 4). Añadir 50 l KH 2 PO 4 por ml de PDT-BzPh, formando una solución micelar nublado. </li><li> Remojar los tubos en la solución PDT-BzPh acidificada durante 40 min a temperatura ambiente en un agitador o en el aparato. </li><li> Enjuagar los tubos con ácido acético diluido (1: 1000) una vez. </li><li> Exponer los tubos a la irradiación UV para una duración total de 60 min. Gire los tubos de ¼ de vuelta cada 15 min para irradiar toda la superficie. </li><li> Remojar los tubos en una solución de 20 mg / ml de bicarbonato de potasio (KHCO3) durante 20 min a temperatura ambiente en un agitador o en el aparato. </li><li> Enjuague los tubos con 3x agua estéril y se almacena a 4 ° C en agua estéril. </li></ol></li><li> Día 3 <ol><li> Disolver 5 mg de sulfo-SMCC en 200 l de dimetilformamida (DMF) (Precaución: Realice en campana de extracción). </li><li> Añadir 50 l de la solución de sulfo-SMCC preparado en la etapa 1 a 0,1 mg / ml de solución de poli-lisina recCD47, y se agita durante 60 min a rotemperatura om. </li><li> Durante 60 minutos de agitación, desgasifique estériles tampón fosfato salino de Dulbecco (DPBS) y reservar. </li><li> Purificar sulfo-SMCC reaccionó recCD47 usando una columna de desalación 7K peso molecular de corte de giro según las instrucciones del fabricante. Recoger el flujo a través de final (de alta calidad recCD47 reactivo con tiol) y diluir a un volumen necesario para recubrir el interior del tubo con DPBS. </li><li> Enjuagar los tubos del día 2 con estéril, desgasificada DPBS 3x. </li><li> Reaccionar la superficie modificada de los tubos con una solución de 20 mg / ml con tris (2-carboxietil) fosfina clorhidrato (TCEP) en DPBS desgasificó durante menos de 2 min. Enjuague los tubos con estéril desgasificado DPBS 4x. </li><li> Añadir la recCD47 reaccionado-sulfo-SMCC a los tubos e incubar durante la noche a 4 ° C en un agitador o en el aparato. </li></ol></li></ol> 2. Inmunoensayo Cuantificación de recCD47 en superficies modificadas <ol><li> Enjuague tubos modificados para ser cuantificado a partir del día 3 ingenioh DPBS 3x. Tubos Store no utilizados para la cuantificación en DPBS a 4 ° C. </li><li> Utilice un punzón de biopsia de 4 mm para hacer muestras de tubos duplicados y colocar golpes de biopsia en pocillos de una placa de 96 pocillos. </li><li> Preparar un control negativo que consiste en un tubo de muestra sin modificar no tratadas con el anticuerpo de detección. Preparar un anticuerpo de control que consiste en un tubo de muestra sin modificar tratado con el anticuerpo de detección. Preparar las muestras de ensayo modificados de tubos tratados con el anticuerpo de detección. </li><li> Muestras de bloque con albúmina de suero bovino 0,4% (BSA) en DPBS durante 60 min a temperatura ambiente en un agitador. </li><li> Después del bloqueo, BSA aspirado y enjuague con solución salina amortiguadora tris más 1% (TBST) 3x-20 Tween, 10 min cada uno en un agitador. </li><li> Prepare una dilución de trabajo del anticuerpo en el 0,4% de BSA de acuerdo a la dilución recomendada por el fabricante para los inmunoensayos. Diluir CD47 humano Anticuerpo B6H12-FITC 1: 100 en 0,4% de BSA. </li><li> Añadir 200 l de dilución de anticuerpo a los pocillos apropiados. Incubacontroles negativos del te con 0,4% sólo BSA. Incubar a temperatura ambiente durante 60 min en un agitador (protegerlo de la luz). </li><li> Enjuague con TBST 3x, 10 min cada uno en un agitador. Aspirar el último aclarado TBST y añadir 200 l DPBS a pocillos de muestras de tubo. </li><li> Recoger el flujo a través final, que es CD47 recombinante reactivo con tiol de alta calidad y diluir a un volumen necesario para recubrir el interior de los tubos con DPBS. </li><li> Leer intensidad de la señal FITC utilizando un lector de microplacas con FITC de excitación (485 nm) y emisión (538 nm) ajustes. </li><li> Calcular recCD47 unido a la superficie polimérica basada en valores estándar, lo que representa para auto-fluorescencia y la unión no específica de la muestra de control del anticuerpo. </li></ol> Protocolo Aparato 3. Chandler Loop Busque la Junta de Revisión Institucional (IRB) la aprobación del protocolo de extracción de sangre e informado consentimiento papeleo antes del inicio de la recolección de muestras de sangre humana. Obtener informaciónrmado el consentimiento de un donante de sangre humana. NOTA: Un diagrama que representa el aparato se muestra en la Figura 2. <ol><li> Llene la bañera con agua destilada hasta aproximadamente media de las ruedas de diámetro están sumergidos. Agregar suficiente cloro para el baño de agua para hacer una solución de lejía al 10%. Establecer baño de agua a 37 ° C y permitir que la temperatura se equilibre. </li><li> Reunir adaptadores de metal (que se muestran en la Figura 1B), sin modificar la tubería y la tubería modificado, y montar alrededor de las ruedas del aparato, como se muestra en la Figura 1C. Asegúrese de que la tubería se ajusta apretadamente alrededor de la rueda con el adaptador de metal en su lugar. </li><li> Obtener 30 ml de muestra de sangre usando un protocolo aprobado por el IRB, en un vial pre-llenado con 2 ml de citrato u otro anticoagulante, y mezclar por inversión para prevenir la coagulación una vez que la muestra se ha recogido. </li><li> Añadir aproximadamente 10 ml de sangre a cada tubo usando la válvula de metal y la jeringa, dejando un poco de aire en el tubo. Asegure la tapa de la válvula y la rOtate durante 3 horas. </li><li> Drenar la sangre en un vaso de residuos tratados con una solución de lejía al 10% (o como lo requiere la política institucional). </li><li> Enjuague suavemente el interior de tubos con DPBS para eliminar todos los rastros de sangre. Recoger el flujo a través en el vaso de precipitados de residuos. Deshágase de la sangre, de acuerdo con la política institucional. </li><li> Desinfectar el aparato y cualquier contacto con la sangre superficies con una solución de lejía al 10% o de acuerdo con la política institucional. </li><li> Procesar muestras para microscopía o microscopía electrónica de barrido fluorescente como se describe a continuación. </li></ol> 4. fluorescente Microscopía y Recuento celular <ol><li> Preparar un 4% de paraformaldehído (PFA) solución (Precaución: Realice en campana de extracción). </li><li> Incubar los tubos en una solución de PFA 4% durante la noche a 4 ° C. </li><li> Después de la incubación durante la noche, quitar 4% PFA y enjuague películas muy suavemente con DPBS. </li><li> Corte el tubo en secciones para exponer la superficie del lumen para la tinción. </li><li> Manchasuna sección de la tubería con unas gotas de medio de montaje con 4 &#39;, 6-diamino-2-fenilindol (DAPI) durante 30 min a temperatura ambiente (proteger de la luz). Después de la tinción, enjuague el tubo muy suavemente con DPBS para reducir el fondo de la señal DAPI. </li><li> Imagen utilizando un microscopio de fluorescencia equipado con un filtro DAPI y la cámara digital para contar el número de células en 9 campos seleccionados a ciegas de vista bajo un aumento de 200X. </li><li> Celular Registros diarios por campo de visión y realizar análisis estadísticos adecuados para determinar la significación estadística de los resultados. </li></ol> 5. Microscopía Electrónica de Barrido <ol><li> Preparar una solución de glutaraldehído al 2% (Precaución: Realice en campana de extracción). </li><li> Incubar los tubos en una solución de glutaraldehído al 2% durante la noche a 4 ° C. </li><li> Después de la incubación durante la noche, lavar suavemente el tubo de 3x con DPBS. </li><li> Prepare una solución al 1% de tetróxido de osmio (Precaución: peligro de inhalación severa! Uso en campana de extracción). </li><li> Incubar los tubos en una solución de 1% de tetróxido de osmio durante 15 min a temperatura ambiente. </li><li> Enjuague suavemente el tubo de 3x con DPBS. </li><li> Preparar una serie de concentraciones de etanol (25%, 50%, 75%, 95% y 100% de etanol). </li><li> Deshidratar el tubo por incubación en la serie de concentraciones de etanol. Incubar en 25%, 50%, 75% y etanol al 95% durante 20 min cada uno. Incubar en etanol al 100% durante 30 min. </li><li> Punto supercrítico seque el tubo con CO 2 durante 45 minutos para eliminar toda la humedad. </li><li> Secciones de tubos de montaje sobre un trozo espécimen con pasta de plata o grafito. </li><li> Secciones de tubo de capa con 12,5 nm de oro-paladio. </li><li> Examinar en el microscopio electrónico de barrido. </li></ol>

<ol>
	<li>Bruck, S. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Medical+applications+of+polymeric+materials.">Medical applications of polymeric materials.</a> Med. Prog. Technol. 9 (1), 1-16 (1982).</li><li>Anderson, J. M., Rodriguez, A., Chang, D. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Foreign+body+reaction+to+biomaterials.">Foreign body reaction to biomaterials.</a> Semin. Immunol. 20 (2), 86-100 (2008).</li><li>Levy, J. H., Tanaka, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inflammatory+response+to+cardiopulmonary+bypass.">Inflammatory response to cardiopulmonary bypass.</a> Ann. Thorac. Surg. 75, S715-S720 (2003).</li><li>Sperling, C., Maitz, M. F., Talkenberger, S., Gouzy, M. F., Groth, T., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+blood+reactivity+to+hydroxylated+and+non-hydroxylated+polymer+surfaces.">In vitro blood reactivity to hydroxylated and non-hydroxylated polymer surfaces.</a> Biomaterials. 28, 3617-3625 (2007).</li><li>Sperling, C., Schweiss, R. B., Streller, U., Werner, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+hemocompatibility+of+self-assembled+monolayers+displaying+various+functional+groups.">In vitro hemocompatibility of self-assembled monolayers displaying various functional groups.</a> Biomaterials. 26, 6547-6457 (2005).</li><li>Vasita, R., Shanmugam, I. K., Katt, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+biomaterials+for+tissue+engineering+applications:+surface+modification+of+polymers.">Improved biomaterials for tissue engineering applications: surface modification of polymers.</a> Curr. Top. Med. Chem. 8, 341-353 (2008).</li><li>Subramanian, S., Parthasarathy, R., Sen, S., Boder, E. T., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Species-+and+cell+type-specific+interactions+between+CD47+and+human+SIRPalpha.">Species- and cell type-specific interactions between CD47 and human SIRPalpha.</a> Blood. 107 (6), 2548-2556 (2006).</li><li>Tsai, R. K., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inhibition+of+'self'+engulfment+through+deactivation+of+myosin-II+at+the+phagocytic+synapse+between+human+cells.">Inhibition of 'self' engulfment through deactivation of myosin-II at the phagocytic synapse between human cells.</a> J Cell Biol. 180 (5), 989-1003 (2008).</li><li>Berg, T. K., vander Schoot, C. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+immune+'self'+recognition:+a+role+for+CD47-SIRPalpha+interactions+in+hematopoietic+stem+cell+transplantation.">Innate immune 'self' recognition: a role for CD47-SIRPalpha interactions in hematopoietic stem cell transplantation.</a> Trends Immunol. 29 (5), 203-206 (2008).</li><li>Oldenborg, P. A., Zheleznyak, A., Fang, Y. F., Lagenaur, C. F., Gresham, H. D., Lindberg, F. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+CD47+as+a+marker+of+self+on+red+blood+cells.">Role of CD47 as a marker of self on red blood cells.</a> Science. 288 (5473), 2051-2054 (2000).</li><li>Stachelek, S. 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=The+effect+of+CD47+modified+polymer+surfaces+on+inflammatory+cell+attachment+and+activation.">The effect of CD47 modified polymer surfaces on inflammatory cell attachment and activation.</a> Biomaterials. 32 (19), 4317-4326 (2001).</li><li>Finley, M. J., Rauva, L., Alferiev, I. S., Weisel, J. W., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diminished+adhesion+and+activation+of+platelets+and+neutrophils+with+CD47+functionalized+blood+contacting+surfaces.">Diminished adhesion and activation of platelets and neutrophils with CD47 functionalized blood contacting surfaces.</a> Biomaterials. 33, 5803-5811 (2012).</li><li>Finley, M. J., Clark, K. A., Alferiev, I. S., Levy, R. J., Stachelek, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intracellular+signaling+mechanisms+associated+with+CD47+modified+surfaces.">Intracellular signaling mechanisms associated with CD47 modified surfaces.</a> Biomaterials. 34, 8640-8649 (2013).</li><li>Ravetch, J. V., Lanier, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immune+inhibitory+receptors.">Immune inhibitory receptors.</a> Science. 290, 84-89 (2000).</li><li>Chandler, A. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+thrombotic+coagulation+of+blood:+a+method+for+producing+a+thrombus.">In vitro thrombotic coagulation of blood: a method for producing a thrombus.</a> Lab Invest. 7, 110-114 (1958).</li><li>Thorsen, T., Klausen, H., Lie, R. T., Holmsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bubble-induced+aggregation+of+platelets:+effects+of+gas+species,+proteins,+and+decompression.">Bubble-induced aggregation of platelets: effects of gas species, proteins, and decompression.</a> Undersea Hyperb Med. 20 (2), 101-119 (1993).</li><li>Ritz-Timme, S., Eckelt, N., Schmidtke, E., Thomsen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genesis+and+diagnostic+value+of+leukocyte+and+platlet+accumulations+around+&#8220;air+bubbles&#8221;+in+blood+after+venous+air+embolism.">Genesis and diagnostic value of leukocyte and platlet accumulations around &#8220;air bubbles&#8221; in blood after venous air embolism.</a> Intl J of Legal Med. 111 (1), 22-26 (1998).</li><li>Miller, R., Fainerman, V. B., W&#252;stneck, R., Kr&#228;gel, J., Trukhin, D. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+the+initial+period+of+protein+adsorption+by+dynamic+surface+tension+measurements+using+different+drop+techniques.">Characterization of the initial period of protein adsorption by dynamic surface tension measurements using different drop techniques.</a> Colloids and Surfaces A. 131 (1-3), 225-230 (1998).</li><li>Oeveren, W. V., Tielliu, I. F., de Hart, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+modified+chandler,+roller+pump,+and+ball+valve+circulation+models+for+in+vitro+testing+in+high+blood+flow+conditions:+application+in+thrombogenicity+testing+of+different+materials+for+vascular+applications.">Comparison of modified chandler, roller pump, and ball valve circulation models for in vitro testing in high blood flow conditions: application in thrombogenicity testing of different materials for vascular applications.</a> Int J Biomater. 2012, (2012).</li></ol>

Las investigaciones realizadas en esta publicación fue apoyada por el Instituto Nacional de Imágenes Biomédicas y Bioingeniería, con el número de adjudicación R21 EB015612 (SSJ), y el Instituto Nacional del Corazón, los Pulmones y la Sangre, con el número de adjudicación T32 HL007915 (JBS y RJL), de la Nacional Instituto de Salud.

La química de fotoactivación (que se resumen en la Figura 1) permite la modificación de prácticamente cualquier superficie de polímero que tiene hidrocarburos suficientes para facilitar PDT-BzPh unión y la posterior irradiación UV para la foto-activar el PDT-BzPh. Funcionalización de la superficie polimérica con grupos tiol reactivos permite la posterior unión de una serie de moléculas de interés comprobables. En nuestros estudios particulares elegimos recombinante CD47 11-13. La ...

Muchos procedimientos clínicos, tales como el bypass cardiopulmonar y la diálisis renal, requieren el uso de conductos de sangre poliméricos y, a menudo están asociados con complicaciones post-procedimiento 1. Cuando perfundido con sangre, estos polímeros ilícito reacción a cuerpo extraño (FBR), que resulta en la adsorción de las proteínas y plaquetas de la sangre, la adhesión de monocitos / macrófagos, y la liberación de citoquinas pro-inflamatorias, todos los cuales contribuyen a complicaciones post-procedimiento y / o 2,3 fallo del dispositivo. Por lo tanto, las estrategias para hacer frente a este problema siguen siendo un área importante de la investigación en curso y los biomateriales. Los investigadores han tratado de abordar este problema mediante la modificación de las superficies de contacto de la sangre con bioactivos o bioinertes moléculas 4-6. La investigación en nuestro laboratorio se ha centrado en añadiendo CD47 recombinante (recCD47) a biomateriales poliméricos como una estrategia para mitigar el FBR y aumentar la eficacia de estos materiales. CD47 es un transmembr de expresión ubicuaane proteína con un papel conocido en la evasión inmune, conceder el carácter de &quot;sí mismo&quot; en las células que expresan 7-10 y se muestra prometedor a conferir biocompatibilidad cuando anexa a superficies poliméricas 11-13. -Señal reguladora proteína alfa (SIRPα), el receptor afín para CD47, y un miembro del motivo inhibidor basado en tirosina inmunoreceptor (ITIM) que contienen familia de proteínas transmembrana, se expresa en células de origen mieloide 14. Anteriormente hemos demostrado que CD47, a través de la señalización celular mediada por SIRPα, abajo-regula la respuesta inmune a poliuretano (PU) y policloruro de vinilo (PVC) en in vitro, ex vivo e in vivo en modelos 11-13. Central con nuestras investigaciones es relativamente nueva química de fotoactivación, descrito en el presente documento, en el que los grupos tiol químicamente reactivos se añaden covalentemente a un tubo polimérico por reacción de la tubería con un polímero multifuncional (PDT-BzPh), compuesto de 2-piridilditio (PDT), la benzofenona fotoreactivo (BzPh) y una polialilamina modificado carboxi-11-13. La reducción de los grupos PDT covalentemente adjuntas con tris (2-carboxietil) fosfina clorhidrato (TCEP) 11 produce una superficie tiolada que puede ser posteriormente se hace reaccionar con restos terapéuticos. Detallada en el presente documento y anteriormente 12,13, recCD47, además modificado con la adición de un poli-lisina C-terminal cola 12,13, se hace reaccionar con sulfosuccinimidilo-4-[N -maleimidomethyl] ciclohexano-1-carboxilato (sulfo-SMCC) durante 1 h para generar grupos reactivos con tiol, lo que permite una formación de enlace monosulfuro entre la tubería y recCD47 11. La capacidad anti-inflamatoria de las superficies funcionalizadas CD47 fue probado, ex viv o, usando el aparato de Chandler Loop con sangre humana total, que fue descrito originalmente en 1958 como un modelo in vitro de la coagulación trombótico 15. El aparato se basa en unasistema de tubo cerrado parcialmente lleno de aire y un motor giratorio para hacer circular la sangre a través del tubo 15. Este modelo experimental ofrece la oportunidad de examinar el efecto de la exposición sangre sobre superficies modificadas y no modificadas, así como el efecto de esas modificaciones de la superficie sobre la fisiología de las células de la sangre. recCD47 se puede añadir a una variedad de superficies poliméricas usando esta química fotoactivación, y su capacidad antiinflamatoria se puede evaluar mediante la utilización de un modelo clínicamente relevante ex vivo imitando perfusión de la sangre sobre superficies poliméricas 11,12. Conductos sanguíneos grado clínico modificados con recCD47 muestran significativamente menos de plaquetas y la fijación de células inflamatorias en comparación con los polímeros no modificados cuando se expone a la sangre humana en el aparato. Una descripción paso a paso de este proceso de modificación se detalla a continuación.

<table border="0" cellpadding="0" cellspacing="0" width="802">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Name</td>
			<td style="width:175px;">Company</td>
			<td style="width:107px;">Catalog Number</td>
			<td style="width:251px;">Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">16% Paraformaldehyde (PFA)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">58906</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">25% Glutaraldehyde</td>
			<td style="width:175px;">VWR</td>
			<td style="width:107px;">AAA17876-AP&#160;</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">2-pyridyldithio,benzophenone (PDT-BzPH)</td>
			<td style="width:175px;">Synthesized in lab</td>
			<td style="width:107px;">N/A</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Bovine Serum Albumin (BSA)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">A3059-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Citrate</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">S5770-50ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Digital Camera</td>
			<td style="width:175px;">Leica</td>
			<td style="width:107px;">DC500</td>
			<td>Out of production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dimethylformamide (DMF)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">270547-100ML</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Dulbecco&#8217;s Phosphate Buffered Saline (DPBS)</td>
			<td style="width:175px;">Gibco/Life Technologies</td>
			<td style="width:107px;">14190-136</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Fluorescent Microscope</td>
			<td style="width:175px;">Nikon</td>
			<td style="width:107px;">TE300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Glacial Acetic Acid</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">A38-212</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Human CD47 (B6H12) &#8211; FITC Antibody</td>
			<td style="width:175px;">Santa Cruz Biotechnology</td>
			<td style="width:107px;">SC-12730</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Osmium Tetroxide</td>
			<td style="width:175px;">Acros Organics</td>
			<td style="width:107px;">197450050</td>
			<td>Caution! Use in fume hood</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Bicarbonate (KHCO3)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">237205-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Potassium Phosphate Monobasic (KH2PO4)</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">P5655-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">PVC Tubing (Cardiovascular Procedure Kit)</td>
			<td style="width:175px;">Terumo Cardiovascular Systems</td>
			<td style="width:107px;">60050</td>
			<td>Most clinical-grade tubing will work</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Scanning Electron Microscope</td>
			<td>JEOL</td>
			<td>JSM-T330A</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Sodium Chloride (NaCl)</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td>BP358-212</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;width:269px;">Microplate Reader</td>
			<td style="width:175px;">Molecular Devices</td>
			<td style="width:107px;">Spectramax Gemini EM&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Sulfo-SMCC</td>
			<td style="width:175px;">Sigma</td>
			<td style="width:107px;">M6035-10MG</td>
			<td>Moisture Sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">tris (2-carboxyethyl) phosphine (TCEP-HCl)</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">20491</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tris Base</td>
			<td style="width:175px;">Sigma</td>
			<td>T1503-100G</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:269px;">Tween-20</td>
			<td>Bio-Rad</td>
			<td>170-6531</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Vectashield with DAPI</td>
			<td style="width:175px;">Fisher Scientific</td>
			<td style="width:107px;">H-1200</td>
			<td>Light sensitive!</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:269px;">Zeba Spin Desalt Columns &#8211; 7K MWCO</td>
			<td style="width:175px;">Thermo Scientific</td>
			<td style="width:107px;">89891</td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of the ex vivo chandler loop apparatus to assess the biocompatibility of modified polymeric blood conduits

La reacción de cuerpo extraño se produce cuando una superficie sintética se introduce en el cuerpo. Se caracteriza por la adsorción de proteínas de la sangre y la posterior unión y activación de las plaquetas, monocitos / macrófagos de adhesión, y los eventos de señalización celular inflamatorios, dando lugar a complicaciones post-procedimiento. El Aparato Chandler Loop es un sistema experimental que permite a los investigadores estudiar las interacciones moleculares y celulares que se producen cuando grandes volúmenes de sangre son perfundidos sobre conductos poliméricos. Para ello, este aparato ha sido usado como un modelo ex vivo permite la evaluación de las propiedades anti-inflamatorias de diversas modificaciones superficie del polímero. Nuestro laboratorio ha demostrado que los conductos sanguíneos, modificados covalentemente a través de la química fotoactivación con CD47 recombinante, pueden conferir biocompatibilidad a superficies poliméricas. Al añadir CD47 a las superficies poliméricas podría ser un medio eficaz para promover la eficacia de los conductos sanguíneos poliméricos. Suein es la metodología que detalla la química de fotoactivación usado para anexar CD47 recombinante a los conductos sanguíneos poliméricos clínicamente relevantes y el uso de la Chandler Loop como modelo experimental ex vivo para examinar las interacciones de la sangre con las CD47 modificado y de control de conductos.

Generación de superficies poliméricos reactivos con tiol mediante el uso de la TFD-BzPh y TCEP junto con reactivo con tiol recCD47 poli-lisina usando SMCC permite la fijación de recCD47 a las superficies poliméricas. El proceso de modificación se resume esquemáticamente en la Figura 1. La conveniencia de este proceso de modificación es que se puede aplicar a muchas proteínas diferentes y muchas superficies poliméricas diferentes, suponiendo que la proteína puede modificarse con grupos química...

Watch this Scientific Journal Video about The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits at JoVE.com

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits.

El uso de la<em&gt; Ex Vivo</em&gt; Aparato Chandler Loop para evaluar la biocompatibilidad de los conductos sanguíneos poliméricos modificados

The foreign body reaction occurs when a synthetic surface is introduced to the body. It is characterized by adsorption of blood proteins and the ...

the-use-ex-vivo-chandler-loop-apparatus-to-assess-biocompatibility

Research

JoVE Journal

Bioengineering

The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

11.5K vistas.  The Children's Hospital of Philadelphia. Blood exposure to polymeric blood conduits initiates the foreign body reaction that has been implicated in clinical complications. Here, the Chandler Loop Apparatus, an experimental tool mimicking blood perfusion through these conduits, is described. Appendage of recombinant CD47 results in decreased evidence of the foreign body reaction on these conduits. 

Video: The Use of the Ex Vivo Chandler Loop Apparatus to Assess the Biocompatibility of Modified Polymeric Blood Conduits

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic valve leaflets are composed of interstitial cells suspended within an extracellular matrix (ECM) and are lined with an endothelial cell monolayer. The valve withstands a harsh, dynamic environment and is constantly exposed to shear, flexion, tension, and compression. Research has shown calcific lesions in diseased valves occur in areas of high mechanical stress as a result of endothelial disruption or interstitial matrix damage1-3. Hence, it is not surprising that epidemiological studies have shown high blood pressure to be a leading risk factor in the onset of aortic valve disease4. 
The only treatment option currently available for valve disease is surgical replacement of the diseased valve with a bioprosthetic or mechanical valve5. Improved understanding of valve biology in response to physical stresses would help elucidate the mechanisms of valve pathogenesis. In turn, this could help in the development of non-invasive therapies such as pharmaceutical intervention or prevention. Several bioreactors have been previously developed to study the mechanobiology of native or engineered heart valves6-9. Pulsatile bioreactors have also been developed to study a range of tissues including cartilage10, bone11 and bladder12. The aim of this work was to develop a cyclic pressure system that could be used to elucidate the biological response of aortic valve leaflets to increased pressure loads. 
The system consisted of an acrylic chamber in which to place samples and produce cyclic pressure, viton diaphragm solenoid valves to control the timing of the pressure cycle, and a computer to control electrical devices. The pressure was monitored using a pressure transducer, and the signal was conditioned using a load cell conditioner. A LabVIEW program regulated the pressure using an analog device to pump compressed air into the system at the appropriate rate. The system mimicked the dynamic transvalvular pressure levels associated with the aortic valve; a saw tooth wave produced a gradual increase in pressure, typical of the transvalvular pressure gradient that is present across the valve during diastole, followed by a sharp pressure drop depicting valve opening in systole. The LabVIEW program allowed users to control the magnitude and frequency of cyclic pressure. The system was able to subject tissue samples to physiological and pathological pressure conditions. This device can be used to increase our understanding of how heart valves respond to changes in the local mechanical environment.

Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

A cyclic pressure bioreactor capable of subjecting heart valve tissue to physiological and pathological pressure conditions has been designed. A LabVIEW program allows users to control pressure magnitude, amplitude and frequency. This device can be used to study the mechanobiology of heart valve tissue or isolated cells.

Diseño de un biorreactor de presión cíclica de la Ex Vivo Estudio de las válvulas aórtica del corazón

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Investigación

Bioingeniería

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced birth defects and many later studies afterwards proved the opposite. Today several harmful xenobiotics like nicotine, heroin, methadone or drugs as well as environmental pollutants were described to overcome this barrier. With the growing use of nanotechnology, the placenta is likely to come into contact with novel nanoparticles either accidentally through exposure or intentionally in the case of potential nanomedical applications. Data from animal experiments cannot be extrapolated to humans because the placenta is the most species-specific mammalian organ 1. Therefore, the ex vivo dual recirculating human placental perfusion, developed by Panigel et al. in 1967 2 and continuously modified by Schneider et al. in 1972 3, can serve as an excellent model to study the transfer of xenobiotics or particles.
Here, we focus on the ex vivo dual recirculating human placental perfusion protocol and its further development to acquire reproducible results.
The placentae were obtained after informed consent of the mothers from uncomplicated term pregnancies undergoing caesarean delivery. The fetal and maternal vessels of an intact cotyledon were cannulated and perfused at least for five hours. As a model particle fluorescently labelled polystyrene particles with sizes of 80 and 500 nm in diameter were added to the maternal circuit. The 80 nm particles were able to cross the placental barrier and provide a perfect example for a substance which is transferred across the placenta to the fetus while the 500 nm particles were retained in the placental tissue or maternal circuit. The ex vivo human placental perfusion model is one of few models providing reliable information about the transport behavior of xenobiotics at an important tissue barrier which delivers predictive and clinical relevant data.

Determination of the Transport Rate of Xenobiotics and Nanomaterials Across the Placenta using the ex vivo Human Placental Perfusion Model

The ex vivo dual recirculating human placental perfusion model can be used to investigate the transfer of xenobiotics and nanoparticles across the human placenta. In this video protocol we describe the equipment and techniques required for a successful execution of a placenta perfusion.

Determinación de la velocidad de transporte de xenobióticos y Nanomateriales través de la placenta mediante el<em&gt; Ex vivo</em&gt; Modelo perfusión placentaria humana

Decades ago the human placenta was thought to be an impenetrable barrier between mother and unborn child. However, the discovery of thalidomide-induced ...

Improved Method for the Preparation of a Human Cell-based, Contact Model of the Blood-Brain Barrier

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has been implied in the etiology of many brain pathologies, creating a need for development of human in vitro BBB models to assist in clinically-relevant research. Among the numerous BBB models thus far described, a static (without flow), contact BBB model, where astrocytes and brain endothelial cells (BECs) are cocultured on the opposite sides of a porous membrane, emerged as a simplified yet authentic system to simulate the BBB with high throughput screening capacity. Nevertheless the generation of such model presents few technical challenges. Here, we describe a protocol for preparation of a contact human BBB model utilizing a novel combination of primary human BECs and immortalized human astrocytes. Specifically, we detail an innovative method for cell-seeding on inverted inserts as well as specify insert staining techniques and exemplify how we use our model for BBB-related research.

Establishment of human models of the blood-brain barrier (BBB) can benefit research into brain conditions associated with BBB failure. We describe here an improved technique for preparation of a contact BBB model, which permits coculturing of human astrocytes and brain endothelial cells on the opposite sides of a porous membrane.

Método mejorado para la preparación de una base de células, Contacto Modelo Humano de la barrera hematoencefálica

The blood-brain barrier (BBB) comprises impermeable but adaptable brain capillaries which tightly control the brain environment. Failure of the BBB has ...

Gene Transfection toward Spheroid Cells on Micropatterned Culture Plates for Genetically-modified Cell Transplantation

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. The cell spheroids are prepared in a culture system on micropatterned plates coated with a thermosensitive polymer. A number of spheroids are formed on the plates, corresponding to the cell adhesion areas of 100 &#181;m diameter that are regularly arrayed in a two-dimensional manner, surrounded by non-adhesive areas that are coated by a polyethylene glycol (PEG) matrix. The spheroids can be easily recovered as a liquid suspension by lowering the temperature of the plates, and their structure is well maintained by passing them through injection needles with a sufficiently large caliber (over 27 G). Genetic modification is achieved by gene transfection using the original non-viral gene carrier, polyplex nanomicelle, which is capable of introducing genes into cells without disrupting the spheroid structure. For primary hepatocyte spheroids transfected with a luciferase-expressing gene, the luciferase is sustainably obtained in transplanted animals, along with preserved hepatocyte function, as indicated by albumin expression. This system can be applied to a variety of cell types including mesenchymal stem cells.

This protocol describes a cell transplantation system using genetically modified, injectable spheroids. Cell spheroids are cultured on micropatterned culture plates and recovered after gene introduction using polyplex nanomicelles. This system facilitates prolonged transgene expression from the transplanted cells in host animals while maintaining the innate function of the cells.

La transfección de genes hacia células Esferoide sobre placas micropatterned Cultura para el trasplante de células genéticamente modificadas

To improve the therapeutic effectiveness of cell transplantation, a transplantation system of genetically modified, injectable spheroids was developed. ...

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential to establish an ex vivo model that can evaluate SIM performance accurately, for developing high-performance SIMs. In our previous study, we developed a new ex vivo model that can be used to evaluate the performance of various SIMs in detail by applying constant tension to the specimen&#39;s ends. We also confirmed that the proposed new ex vivo model allows accurate submucosal elevation height (SEH) measurement under uniform conditions and detailed comparisons of the performances of various types of SIMs. Here, we describe the new ex vivo model and explain the detailed setup methodology of this model. Since all parts of the new model were easy to obtain, the setup of the new model could be completed quickly. SEH of various SIMs could be measured more accurately by using the new model. The critical factor that determines SIM performance can be identified using the new model. SIM development speed will drastically increase after the factor has been identified.

A New Ex Vivo Model for the Evaluation of Endoscopic Submucosal Injection Material Performance

We developed a new ex vivo model that applies constant tension to the porcine gastric specimen. This development made it possible to evaluate the performance (the height and duration of the submucosal elevation) of various SIMs accurately.&#160; The detailed setup methodology of this new model is explained.

Un nuevo modelo Ex Vivo para la evaluación de la ejecución Material de la inyección Submucosal endoscópica

Increasing the performance of submucosal injection materials (SIMs) is important for endoscopic therapy of early gastrointestinal cancer. It is essential ...

Intra-cardiac Side-Firing Light Catheter for Monitoring Cellular Metabolism using Transmural Absorbance Spectroscopy of Perfused Mammalian Hearts

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome absorbance respectively. In addition, numerous aspects of the mitochondrial metabolic status such as membrane potential and substrate entry can also be estimated. To perform cardiac wall transmission optical spectroscopy, a commercially available side-firing optical fiber catheter is placed in the left ventricle of the isolated perfused heart as a light source. Light passing through the heart wall is collected with an external optical fiber to perform optical spectroscopy of the heart in near real- time. The transmission approach avoids numerous surface scattering interference occurring in widely used reflection approaches. Changes in transmural absorbance spectra were deconvolved using a library of chromophore reference spectra, providing quantitative measures of all the known cardiac chromophores simultaneously. This spectral deconvolution approach eliminated intrinsic errors that may result from using common dual wavelength methods applied to overlapping absorbance spectra, as well as provided a quantitative evaluation of the goodness of fit. A custom program was designed for data acquisition and analysis, which permitted the investigator to monitor the metabolic state of the preparation during the experiment. These relatively simple additions to the standard heart perfusion system provide a unique insight into the metabolic state of the heart wall in addition to conventional measures of contraction, perfusion, and substrate/oxygen extraction.

Here we introduce a method for using an intra-ventricle optical catheter in perfused hearts to perform absorbance spectroscopy across the heart wall. The data obtained provides robust information on tissue oxygen tension as well as substrate utilization and membrane potential simultaneously with cardiac performance measures in this ubiquitous preparation.

Catéter de luz intracardiaca para monitorear el metabolismo celular utilizando espectroscopia de absorbancia transmural de corazones de mamíferos perfundidos

Absorbance spectroscopy of cardiac muscle provides non-destructive assessment of cytosolic and mitochondrial oxygenation via myoglobin and cytochrome ...

Blood Flow Imaging with Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler modes, it has several limits. Firstly, a finely tuned signal filtering operation is needed to distinguish blood flows from surrounding moving tissues. Secondly, the operator must choose between localizing the blood flows or quantifying them. In the last two decades, ultrasound imaging has undergone a paradigm shift with the emergence of ultrafast ultrasound using unfocused waves. In addition to a hundredfold increase in framerate (up to 10000 Hz), this new technique also breaks the conventional quantification/localization trade-off, offering a complete blood flow mapping of the field of view and a simultaneous access to fine velocities measurements at the single-pixel level (down to 50 &#181;m). This data continuity in both spatial and temporal dimensions strongly improves the tissue/blood filtering process, which results in an increase sensitivity to small blood flow velocities (down to 1 mm/s). In this method paper, we aim to introduce the concept of ultrafast Doppler as well as its main parameters. Firstly, we summarize the physical principles of unfocused wave imaging. Then, we present the Doppler signal processing main steps. Particularly, we explain the practical implementation of the critical tissue/blood flow separation algorithms and on the extraction of velocities from these filtered data. This theoretical description is supplemented by in vitro experiences. A tissue phantom embedding a canal with flowing blood-mimicking fluid is imaged with a research programmable ultrasound system. A blood flow image is obtained and the flow characteristics are displayed for several pixels in the canal. Finally, a review of in vivo applications is proposed, showing examples in several organs such as carotids, kidney, thyroid, brain and heart.

This protocol shows how to apply ultrafast ultrasound Doppler imaging to quantify blood flows. After a 1 s long acquisition, the experimenter has access to a movie of the full field of view with axial velocity values for each pixel every &#8776;0.3 ms (depending on the ultrasound time of flight).

Imágenes de flujo sanguíneo con Ultrafast Doppler

The pulsed-Doppler effect is the main technique used in clinical echography to assess blood flow. Applied with conventional focused ultrasound Doppler ...

&quot;Avatar&quot;- Optimized Ex Vivo Work Loop Experiment to Emulate In Vivo Muscle Force

"Avatar", a Modified Ex vivo Work Loop Experiments Using In vivo Strain and Activation

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and mechanical systems occurs at all levels of biological organization concurrently, from the tuning of leg muscle properties while running to the dynamics of the limbs interacting with the ground. Understanding the conditions under which animals shift their neural control strategies toward intrinsic muscle mechanics (&#39;preflexes&#39;) in the control hierarchy would allow muscle models to predict in vivo muscle force and work more accurately. To understand in vivo muscle mechanics, ex vivo investigation of muscle force and work under dynamically varying strain and loading conditions similar to in vivo locomotion is required. In vivo strain trajectories typically exhibit abrupt changes (i.e., strain and velocity transients) that arise from interactions among neural activation, musculoskeletal kinematics, and loads applied by the environment. The principal goal of our &#34;avatar&#34; technique is to investigate how muscles function during abrupt changes in strain rate and loading when the contribution of intrinsic mechanical properties to muscle force production may be highest. In the &#34;avatar&#34; technique, the traditional work-loop approach is modified using measured in vivo strain trajectories and electromyographic (EMG) signals from animals during dynamic movements to drive ex vivo muscles through multiple stretch-shortening cycles. This approach is similar to the work-loop technique, except that in vivo strain trajectories are scaled appropriately and imposed on ex vivo mouse muscles attached to a servo motor. This technique allows one to: (1) emulate in vivo strain, activation, stride frequency, and work-loop patterns; (2) vary these patterns to match in vivo force responses most accurately; and (3) vary specific features of strain and/or activation in controlled combinations to test mechanistic hypotheses.

Author Spotlight: Bridging the Gap Between In Vivo and Ex Vivo Studies with the &quot;Avatar&quot; Technique to Advance Muscle Mechanics Research 

This article details the methodology for emulating in vivo muscle force production during ex vivo work loop experiments using an "avatar" muscle from a laboratory rodent to assess the contributions of strain transients and activation to the muscle force response.

Movement behaviors are emergent features of dynamic systems that result from muscle force production and work output. The interplay between neural and ...

Preparation of Cardiomyocytes and Setting-Up the Apparatus for the Slow Injection Method

Prepare the 10%gelatin stock solution by weighing and dissolving the gelatin in the ADS buffer, and autoclaving the solution. Rewarm the 10%gelatin stock at 37 degrees Celsius, until it becomes liquid. Then dilute the gelatin stock, using a prewarmed ADS buffer, to obtain the final injectable 5%gelatin solution.Suspend 96 cardiomyocyte balls in 100 microliters of prewarmed injection solution. Load the cardiomyocyte ball suspension into the cell injection syringe without aspirating the excess air. Next, hold the syringe vertically, with the needle facing upward, and tap the syringe to collect the bubbles at the upper ridge of the syringe.Also, observe the cardiomyocyte balls gradually settling onto the rubber seal of the plunger. Maintain the vertical position of the syringe, and slowly push the plunger to discard the bubbles and excess cell suspension solution, until 20 microliters of the cell suspension remain in the syringe, and the cardiomyocyte ball remains resting on the rubber seal of the plunger. Once done, immerse the capped syringe in an ice bath for five minutes, and fix the injection syringe apparatus on the animal stage before activating the main syringe pump during percutaneous cell transplantation in an anesthetized rat.

Preparación de cardiomiocitos y puesta a punto del aparato para el método de inyección lenta

Análisis de modelos de barrera hematoencefálica mediante tinción de inmunofluorescencia

Use of the MicroSiM (&#181;SiM) Barrier Tissue Platform for Modeling the Blood-Brain Barrier

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, the&#160;&#181;SiM provides experimentalists with new capabilities, including live cell imaging, unhindered paracrine signaling between &#39;blood&#39; and &#39;brain&#39; chambers, and the ability to directly image immunofluorescence without the need for the extraction/remounting of membranes. Here we demonstrate the basic use of the platform to establish monoculture (endothelial cells) and co-culture (endothelial cells and pericytes) models of the BBB using ultrathin nanoporous silicon-nitride membranes. We demonstrate compatibility with both primary cell cultures and human induced pluripotent stem cell (hiPSC) cultures. We provide methods for qualitative analysis of BBB models via immunofluorescence staining and demonstrate the use of the &#181;SiM for the quantitative assessment of barrier function in a small molecule permeability assay. The methods provided should enable users to establish their barrier models on the platform, advancing the use of tissue chip technology for studying human tissues.

Autor destacado: Avanzando en el uso de la tecnología de chips de tejido para el estudio de tejidos humanos 

This report provides protocols for assembly, cell culture, and assays on the &#181;SiM platform for the construction of blood-brain barrier models.

Uso de la plataforma de tejido de barrera MicroSiM (μSiM) para modelar la barrera hematoencefálica

The microSiM (&#181;SiM) is a membrane-based culture platform for modeling the blood-brain barrier (BBB). Unlike conventional membrane-based platforms, ...