Este estudio se realizó en el programa de investigación de los materiales biomédicos (BMM) instituto. BMM está cofinanciado por el Ministerio de Asuntos Económicos, Agricultura e Innovación holandés. La contribución financiera de la Hartstichting Nederlandse se agradece.

1. Cultura de la vena safena Humanos Células <ol><li> Aislar las células de la vena safena magna, adquirido a partir de un donante, de conformidad con las directrices para el material de uso secundario, de acuerdo con el protocolo por Schnell et al. 9 y almacenar estos en nitrógeno líquido. Desde la parte de la vena safena magna de uno piezas cortadas donantes de 2 x 2 mm a la cultura en una placa de seis pocillos. Utilice 2 piezas por pocillo. Suficientes células pueden obtenerse generalmente para llenar cerca de 3 viales de 0.25 x 10 6 células en nitrógeno líquido. HVSCs se caracterizan como miofibroblastos, por que muestra la expresión de vimentina, ninguna expresión de desmina y una subpoblación expresar α actina del músculo liso 10. Siguiente iniciar el protocolo de descongelar las células del nitrógeno líquido para aumentar el número de células. </li><li> Colocar las células de un vial en un matraz de cultivo de tejido T75 y añadir medio de crecimiento (GM), que consta de avanzada DMEM, 50 ml de suero de bovino fetal (FBS), 5 ml de penicilina /estreptomicina y 5 ml de L-glutamax. Cambie medio cada 2-3 días. </li><li> Las células crecen confluente aproximadamente después de 14 días. Tienda 0.5 x 10 6 células en un vial en nitrógeno líquido, denominado paso 1. </li></ol> Nota: La congelación de las células no es necesario cuando la cosecha HVSCs, pero se utiliza únicamente para el almacenamiento. <ol start="4"><li> Coloque los HVSCs de un vial en un frasco de cultivo de tejidos T175 y añadir GM. Cambie medio cada 2-3 días. Las células crecen confluente después de aproximadamente 7 días. Tienda 3 x 10 6 células en un vial en nitrógeno líquido, denominado paso 2. </li><li> Coloque las células de un vial en una rollerbottle y añade GM. Cambie medio cada 2-3 días. Las células crecen confluente aproximadamente después de 8 días. Una rollerbottle contiene aproximadamente 20 a 30 x 10 6 células. Cultura células hasta el paso 6. No utilice las células de un pasaje más altos que el paso 9, ya que el fenotipo de células puede cambiar. </li></ol&gt; 2. Ingeniería de construcciones de tejido a base de fibrina <ol><li> Preparar pegamento de silicona mezclando el elastómero con el agente de curado (10:01). Cortar 7 x 3 segmentos rectangulares mm de Velcro. Pegue el velcro en una placa de cultivo de 6 pocillos, con membranas flexibles para formar una cruz, y dejar un espacio cuadrado de 3 mm entre las tiras de velcro. </li></ol> Notas: Utilice sólo el lado suave del velcro y se enfrentan a este lado hacia arriba. Al pegar el velcro, sólo cubren el velcro con pegamento de silicona, no se extendió en todo el pegamento también. Dado que las placas de cultivo tienen fondos de membrana de silicona, usar algo por debajo de la placa de refuerzo para las membranas flexibles, para garantizar un fácil pegado en la placa. <ol start="2"><li> Se seca el pegamento de silicona durante la noche en un horno a 60 ° C para asegurar el endurecimiento del pegamento. Esterilizar mediante la adición de EtOH al 70% a los pocillos y se incuba durante 30 min. Lavar 3 veces con PBS y PBS de eliminar todo el WELls y el velcro. Poner bajo UV durante 30 min y mantener estéril hasta su uso. </li><li> Para preparar el medio de Ingeniería de Tejidos (TM), que consiste en GM y 130 mg de ácido L-ascórbico 2-fosfato. </li><li> Añadir 1 mg / ml de ácido caproico ε-amino (ACA) de la TM. ACA se usa durante los primeros 7 días de cultivo para evitar que la fibrina se degrade 11. Alternativamente, se puede utilizar aprotinina. </li><li> Para evitar la formación de grupo, permite fibrinógeno alcance la temperatura ambiente antes de abrirlo. Sin grumos fibrinógeno se disolverá más fácilmente. Disolver el fibrinógeno en una concentración de 10 mg real de proteína / ml TM 7 suplementado con ACA. La solución de fibrinógeno, podría ser necesaria filtro estéril varios filtros. Almacenar en hielo hasta su uso. </li></ol> Nota: Para disolver la mezcla de fibrinógeno suavemente para evitar la formación de espuma demasiado. <ol start="6"><li> Disolver la trombina a una concentración de 10 UI / ml TM 7 suplementado con ACA. Almacenar en hielo hasta su uso. </li> &lt;/ Ol&gt; Nota: se necesita almacenamiento en hielo para evitar la gelificación inicial de trombina y fibrinógeno. <ol start="7"><li> Tripsinizar las células, resuspender en GM y el recuento. </li><li> Usa 15 x 10 6 células / ml para la siembra de las construcciones de tejido a base de fibrina (concentración basada en métodos para válvulas cardíacas de ingeniería de tejidos 7). Un único gel consiste en 100 l gof el, y por lo tanto de 1,5 x 10 6 células. Ponga 10 6 células de 1,5 x en un tubo de centrífuga, lo que resulta en el mayor número tubos de centrífuga como el número de geles que se harán. </li><li> Centrifugar las células a 350 xg durante 7 minutos y desechar el sobrenadante. Resuspender las células en 50 l de trombina. Añadir 4 l de microesferas de poliestireno azul fluorescente a esta suspensión. Ponga 50 l de fibrinógeno en un vial. Utilice una pipeta para tomar la 50 l de trombina con las células. Aumentar el volumen de la pipeta a 100 l. Pipetear 50 l de la solución de trombina conlas células en el vial con fibrinógeno para mezclar la trombina y el fibrinógeno, y ocupan la mezcla de 100 l. </li></ol> Nota: El fluorescente microesferas de poliestireno se utilizan como marcadores de referencia internos para el análisis de imagen. Cuando la mezcla de trombina y fibrinógeno, previenen la formación de burbujas de aire por pipeteo cuidadosamente la mezcla. Las burbujas de aire se traducirá en agujeros en el gel de fibrina. <ol start="10"><li> Pipetear la mezcla de gel en y entre las tiras de Velcro. El tiempo de gelificación típico de los geles de fibrina, una vez que los componentes se mezclan, es del orden de 20 seg. </li></ol> Notas: Para ello, tan pronto como sea posible para evitar la gelificación antes de que la mezcla se pipeteó en la placa de pocillos. Practique antes de utilizar las células y los granos. <ol start="11"><li> Incubar suspensiones para 30 min a 37 ° C en un humidificado 95% de aire / 5% de CO 2 incubadora para permitir la gelificación antes de añadir la cultura TM con ACA. </li><li> Reemplace TM con ACA cada 2-3 días para los primeros 7 días. Los geles son lo suficientemente estable después de una semana para ser cultivado sin ACA. Después de la primera semana reemplazar TM cada 2-3 días. Añadir 6 ml de TM por pocillo. El día 12 células han producido suficiente colágeno para la visualización. </li></ol> 3. La aplicación de la tensión y la inducción de cambios en la cepa y limitaciones <ol><li> Cepa estático se aplica directamente por las células, debido a la tracción celular y compactación 12. Para inducir la reorganización de colágeno, cortar el tejido construir suelta de dos tiras de Velcro en una dirección. Esto se traduce en limitaciones unidireccionales (Figura 1A). Realice esto en 12 días, ya que el constructo es entonces lo suficientemente estable y el colágeno se ha depositado. </li><li> Aplicar tensión cíclica mediante la aplicación de un vacío a la parte inferior de las placas de cultivo con membranas flexibles, con el uso del sistema de FX4000T Flexcell. Coloque las placas en una placa de base, en la parte superior de los puestos de carga (que son una parte de thdispositivo de carga cíclica e). La bomba se aplica un vacío sobre la membrana y por lo tanto tira de la membrana sobre el poste rectangular, se extiende por debajo. Debido a los archivos adjuntos en forma de cruz del tejido construye a la membrana (a través de la velcro) y el poste rectangular de la cepa cíclico aplicado es uniaxial (Figura 1B). </li><li> Inicialmente, utilizar cultivo estático durante 5 días para lograr la integridad mecánica inicial, antes de la aplicación de la cepa cíclico a partir del día 6. Programa de un protocolo de tinción cíclico en el controlador para la bomba de vacío. Por ejemplo, usar un protocolo de tensión cíclica intermitente previamente establecido, con la dirección uniaxial 13. Este consiste en una cepa de intermitente de una onda sinusoidal con 1 Hz, el esfuerzo de 0 a 5% de deformación, por períodos de 3 hr, 3 hr alternadas con periodos de descanso. Lleve a cabo este protocolo de tensión cíclica durante 7 días, la inducción de una organización de colágeno alineadas. </li><li> Normalmente, después de 12 días HVSCs han producido un organizatio colágeno alineadasn. Después de que se alcanza una organización de colágeno alineados, cambiar la dirección de la tensión cíclica uniaxial, para ser perpendicular a la dirección cepa original. Para ello, girar los postes rectangulares de 90 °. </li></ol> 4. Visualización de las células y el colágeno <ol><li> Para visualizar activo, la remodelación del colágeno en tiempo real, muestras de etiquetas con sondas que no interfieren con la viabilidad celular o la formación de colágeno. Usar sondas para teñir el citoplasma de la célula fluorescente y el colágeno. </li><li> Alternativamente segunda generación de armónicos usando microscopía confocal de barrido láser se puede utilizar para visualizar las estructuras de colágeno utilizando autofluorescencia 14, con excitación a 780 nm y la detección entre 500-550 nm. </li><li> Eliminar las placas de cultivo de la configuración y la incubadora, para las muestras en función del ciclo tensas durante el período de descanso de 3 horas, y transportarlos a un microscopio de escaneo láser confocal para visualizar las células y el colágeno. </li></ol>

<ol>
	<li>Beamish, J. A., He, P., Kottke-Marchant, K., Marchant, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+regulation+of+contractile+smooth+muscle+cell+phenotype:+implications+for+vascular+tissue+engineering.">Molecular regulation of contractile smooth muscle cell phenotype: implications for vascular tissue engineering.</a> Tissue Eng. Part B Rev. 16, 467-491 (2010).</li><li>Isenberg, B. C., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+cyclic+distention+enhances+the+mechanical+properties+of+collagen-based+media-equivalents.">Long-term cyclic distention enhances the mechanical properties of collagen-based media-equivalents.</a> Ann. Biomed. Eng. 31, 937-949 (2003).</li><li>Nichol, J. W., Khan, A. R., Birbach, M., Gaynor, J. W., Gooch, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hemodynamics+and+axial+strain+additively+increase+matrix+remodeling+and+MMP-9,+but+not+MMP-2,+expression+in+arteries+engineered+by+directed+remodeling.">Hemodynamics and axial strain additively increase matrix remodeling and MMP-9, but not MMP-2, expression in arteries engineered by directed remodeling.</a> Tissue Eng. Part A. 15, 1281-1290 (2009).</li><li>Sander, E. A., Stylianopoulos, T., Tranquillo, R. T., Barocas, V. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Image-based+multiscale+modeling+predicts+tissue-level+and+network-level+fiber+reorganization+in+stretched+cell-compacted+collagen+gels.">Image-based multiscale modeling predicts tissue-level and network-level fiber reorganization in stretched cell-compacted collagen gels.</a> Proc. Natl. Acad. Sci. U.S.A. 106, 17675-17680 (2009).</li><li>Hu, J. J., Humphrey, J. D., Yeh, A. T. <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+engineered+tissue+development+under+biaxial+stretch+using+nonlinear+optical+microscopy.">Characterization of engineered tissue development under biaxial stretch using nonlinear optical microscopy.</a> Tissue Eng. Part A. 15, 1553-1564 (2009).</li><li>Lee, E. J., Holmes, J. W., Costa, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remodeling+of+engineered+tissue+anisotropy+in+response+to+altered+loading+conditions.">Remodeling of engineered tissue anisotropy in response to altered loading conditions.</a> Ann. Biomed. Eng. 36, 1322-1334 (2008).</li><li>Mol, A., 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=Fibrin+as+a+cell+carrier+in+cardiovascular+tissue+engineering+applications.">Fibrin as a cell carrier in cardiovascular tissue engineering applications.</a> Biomaterials. 26, 3113-3121 (2005).</li><li>de Jonge, N., Kanters, F. M., Baaijens, F. P., Bouten, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41 (4), 763-774 (2012).</li><li>Schnell, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimal+cell+source+for+cardiovascular+tissue+engineering:+venous+vs.+aortic+human+myofibroblasts.">Optimal cell source for cardiovascular tissue engineering: venous vs. aortic human myofibroblasts.</a> Thorac. Cardiovasc. Surg. 49, 221-225 (2001).</li><li>Mol, A., 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=Autologous+human+tissue-engineered+heart+valves:+prospects+for+systemic+application.">Autologous human tissue-engineered heart valves: prospects for systemic application.</a> Circulation. , I152-I158 (2006).</li><li>Ahmann, K. A., Weinbaum, J. S., Johnson, S. L., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibrin+degradation+enhances+vascular+smooth+muscle+cell+proliferation+and+matrix+deposition+in+fibrin-based+tissue+constructs+fabricated+in+vitro.">Fibrin degradation enhances vascular smooth muscle cell proliferation and matrix deposition in fibrin-based tissue constructs fabricated in vitro.</a> Tissue Eng. Part A. 16, 3261-3270 (2010).</li><li>John, J., Quinlan, A. T., Silvestri, C., Billiar, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Boundary+stiffness+regulates+fibroblast+behavior+in+collagen+gels.">Boundary stiffness regulates fibroblast behavior in collagen gels.</a> Ann. Biomed. Eng. 38, 658-673 (2010).</li><li>Rubbens, M. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intermittent+straining+accelerates+the+development+of+tissue+properties+in+engineered+heart+valve+tissue.">Intermittent straining accelerates the development of tissue properties in engineered heart valve tissue.</a> Tissue Eng. Part A. 15, 999-1008 (2009).</li><li>Chen, W. L., 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=Multiphoton+imaging+and+quantitative+analysis+of+collagen+production+by+chondrogenic+human+mesenchymal+stem+cells+cultured+in+chitosan+scaffold.">Multiphoton imaging and quantitative analysis of collagen production by chondrogenic human mesenchymal stem cells cultured in chitosan scaffold.</a> Tissue Eng. Part C Methods. 16, 913-920 (2010).</li><li>de Jonge, N., Kanters, F. M., Baaijens, F. P., Bouten, C. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-induced+Collagen+Organization+at+the+Micro-level+in+Fibrin-based+Engineered+Tissue+Constructs.">Strain-induced Collagen Organization at the Micro-level in Fibrin-based Engineered Tissue Constructs.</a> Ann. Biomed. Eng. 41, 763-774 (2013).</li><li>Merryman, W. D., 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=Correlation+between+heart+valve+interstitial+cell+stiffness+and+transvalvular+pressure:+implications+for+collagen+synthesis.">Correlation between heart valve interstitial cell stiffness and transvalvular pressure: implications for collagen synthesis.</a> Am. J. Physiol. 290, (2006).</li><li>Ingber, 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=From+cellular+mechanotransduction+to+biologically+inspired+engineering:+2009+Pritzker+Award+Lecture,+BMES+Annual+Meeting+October+10,+2009.">From cellular mechanotransduction to biologically inspired engineering: 2009 Pritzker Award Lecture, BMES Annual Meeting October 10, 2009.</a> Ann. Biomed. Eng. 38, 1148-1161 (2009).</li><li>Sander, E. A., Barocas, V. H., Tranquillo, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+fiber+alignment+pattern+alters+extracellular+matrix+synthesis+in+fibroblast-populated+fibrin+gel+cruciforms+and+correlates+with+predicted+tension.">Initial fiber alignment pattern alters extracellular matrix synthesis in fibroblast-populated fibrin gel cruciforms and correlates with predicted tension.</a> Ann. Biomed. Eng. 39, 714-729 (2010).</li><li>van der Schaft, D. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+Skeletal+Muscle+Tissues+from+Murine+Myoblast+Progenitor+Cells+and+Application+of+Electrical+Stimulation.">Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation.</a> J. Vis. Exp. (73), e4267 (2013).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

El sistema modelo descrito de construcciones de fibrina por células pobladas tiene un gran potencial para el estudio de células y colágeno (re) organización (de Jonge et al. 15), por ejemplo, para ser utilizado para los propósitos de ingeniería de tejidos. Mediante el uso de fibrina como el portador inicial de la célula, después de la degradación de fibrina, se crea un tejido con las células y la matriz sólo endógena. De esta manera, las células son estimuladas para reaccionar a...

Tejidos cardiovasculares tienen una función de soporte importante. En particular, el contenido y la organización de las fibras de colágeno en la matriz extracelular contribuir a las propiedades de carga y dominar a la fuerza general del tejido 1. En la ingeniería de tejidos se utiliza acondicionado mecánico de la construcción - por lo general consiste en (cíclico) regímenes tensos - para mejorar la organización del tejido y las propiedades mecánicas 2,3. Aún no se ha logrado la comprensión completa de la organización de colágeno inducida por deformación en geometrías complejas de tejidos para crear tejidos con colágeno arquitectura predefinida. Esto se debe principalmente a nuestro conocimiento limitado de la remodelación del colágeno en los tejidos en desarrollo. Los modelos existentes dan principalmente información sobre el resultado neto final de la remodelación del colágeno con el uso de la tensión estática 4-6. Aquí, se proporciona un sistema modelo altamente ajustable que permite el estudio de la organización de colágeno (re) de una manera en tiempo real, en 3D, bajo la influenciade tensión estática o cíclico. Las construcciones son de tejido a base de fibrina, asegurarse de que todas las colágeno en la construcción es endógeno. Células y colágeno organización en las construcciones se visualiza, y un sistema de referencia interna nos permite reubicar las células y las estructuras de colágeno para un análisis cronológico. En este protocolo se describe el uso del sistema de modelo para células Vena safena Humanos (HVSCs), ya que estas células son conocidos por su aumento de la producción de matriz extracelular y la capacidad de remodelar la matriz y el uso establecido en los tejidos cardiovasculares de ingeniería 7, basado en la obra de De Jonge et al. 8

<table border="1">
	<tbody>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Culture plastic</td>
			<td>Greiner</td>
			<td></td>
			<td>Includes culture flasks and pipettes</td>
		</tr>
		<tr>
			<td>Advanced DMEM</td>
			<td>Gibco</td>
			<td>12491</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Greiner</td>
			<td>758075</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/streptomycin</td>
			<td>Gibco</td>
			<td>10378016</td>
			<td></td>
		</tr>
		<tr>
			<td>GlutaMax</td>
			<td>Gibco</td>
			<td>35050-079</td>
			<td></td>
		</tr>
		<tr>
			<td>Elastomer and curing agent</td>
			<td>Dow Corning Corporation</td>
			<td>3097358-1004</td>
			<td>Silastic MDX 4-4210#</td>
		</tr>
		<tr>
			<td>Velcro</td>
			<td>Regular store</td>
			<td></td>
			<td>You can buy this at a regular store, only use the soft side</td>
		</tr>
		<tr>
			<td>Bioflex culture plates</td>
			<td>Flexcell Int</td>
			<td>BF-3001U</td>
			<td>Untreated</td>
		</tr>
		<tr>
			<td>L-Ascorbic Acid 2-phosphatase</td>
			<td>Sigma</td>
			<td>A8960</td>
			<td></td>
		</tr>
		<tr>
			<td>&#949;-Amino Caproic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>D7754</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine thrombin</td>
			<td>Sigma</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine fibrinogen</td>
			<td>Sigma</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr>
			<td>0.45 syringe filter</td>
			<td>Whatmann (Schleicher and Scheul)</td>
			<td>10462100</td>
			<td></td>
		</tr>
		<tr>
			<td>Polystyrene microspheres</td>
			<td>Invitrogen</td>
			<td>F-8829</td>
			<td>Blue fluorescent, 10 &#956;m diameter</td>
		</tr>
		<tr>
			<td>Flexcell FX-4000T</td>
			<td>Flexcell Int</td>
			<td></td>
			<td>Includes rectangular loading posts</td>
		</tr>
		<tr>
			<td>Cell Tracker Orange</td>
			<td>Invitrogen Molecular Probes</td>
			<td>C2927</td>
			<td></td>
		</tr>
		<tr>
			<td>CNA35-OG488</td>
			<td></td>
			<td></td>
			<td>Cordially provided by the Laboratory for Macromolecular and Organic Chemistry, Department of Biomedical Engineering, Eindhoven University of Technology</td>
		</tr>
		<tr>
			<td>Confocal laser scanning microscope</td>
			<td>Carl Zeiss</td>
			<td></td>
			<td>LSM 510 Meta laser scanning microscope and Two-Photon-LSM mode</td>
		</tr>
		<tr>
			<td>Amphotericin</td>
			<td>Gibco</td>
			<td>15290-018</td>
			<td>Needed for cell isolation</td>
		</tr>
	</tbody>
</table>

engineering fibrin-based tissue constructs from myofibroblasts and application of constraints and strain to induce cell and collagen reorganization

Contenido de colágeno y la organización en el desarrollo de tejidos de colágeno pueden ser influenciados por las cepas locales del tejido y tejido de restricción. Ingenieros de tejidos pretenden utilizar estos principios para crear tejidos con colágeno arquitecturas predefinidas. Una comprensión completa de los procesos subyacentes exactas de la remodelación del colágeno para controlar la arquitectura final del tejido, sin embargo, es insuficiente. En particular, se sabe poco acerca de la orientación de la (re) producción de fibras de colágeno en respuesta a cambios en las condiciones de carga mecánica del tejido. Hemos desarrollado un sistema modelo in vitro, que consiste en biaxialmente con limitaciones de miofibroblastos cabezas de serie de fibrina construcciones, para aclarar aún más colágeno (re) orientación en respuesta a i) revertir biaxial a condiciones de carga estática uniaxiales y ii) cíclico carga uniaxial de la biaxialmente limitado construcciones antes y después de un cambio en la dirección de carga, con el uso del dispositivo de carga FX4000T Flexcell. Time-lapse confocal de imágenes se utiliza a visualize orientación colágeno (re) de una manera no destructiva. Células y colágeno organización en las construcciones se puede visualizar en tiempo real, y un sistema de referencia interna nos permite reubicar las células y las estructuras de colágeno para un análisis cronológico. Varios aspectos del sistema de modelo se pueden ajustar, como fuente de células o el uso de células sanas y enfermas. Los aditivos pueden ser utilizados para aclarar aún más los mecanismos de remodelación del colágeno subyacente, por ejemplo la adición de las MMPs o el bloqueo de las integrinas. Forma y tamaño de la construcción se pueden adaptar fácilmente a las necesidades específicas, lo que resulta en un sistema modelo altamente ajustable para estudiar la organización de células y colágeno (re).

Este sistema modelo permite para el cultivo de miofibroblastos de semilla geles de fibrina. Figura 1A muestra un tejido cultivado primera virtud de las limitaciones biaxial estáticas. Limitaciones de tejido son liberados por corte del gel de fibrina a partir de dos restricciones, para crear restricciones estáticos uniaxiales, y compacta de tejido y remodela después (Figura 1A). Para cepa cíclico, el tejido se cultiva bajo limitaciones biaxial estáticas así. Después de 5 días ten...

Watch this Scientific Journal Video about Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization at JoVE.co

Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Este sistema modelo se inicia a partir de un gel de fibrina-miofibroblastos poblado que se puede utilizar para estudiar colágeno endógeno (re) organización en tiempo real de una manera no destructiva. El modelo de sistema es muy sintonizable, ya que se puede utilizar con diferentes fuentes de células, aditivos de medio, y se puede adaptar fácilmente a las necesidades específicas.

Ingeniería fibrina basado en construcciones de tejido de miofibroblastos y de aplicación de las restricciones y la tensión para inducir la organización de células y colágeno (Re)

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim ...

engineering-fibrin-based-tissue-constructs-from-myofibroblasts

Research

JoVE Journal

Bioengineering

10.8K vistas.  Eindhoven University of Technology.  Este sistema modelo se inicia a partir de un gel de fibrina-miofibroblastos poblado que se puede utilizar para estudiar colágeno endógeno (re) organización en tiempo real de una manera no destructiva. El modelo de sistema es muy sintonizable, ya que se puede utilizar con diferentes fuentes de células, aditivos de medio, y se puede adaptar fácilmente a las necesidades específicas. 

Video: Engineering Fibrin-based Tissue Constructs from Myofibroblasts and Application of Constraints and Strain to Induce Cell and Collagen Reorganization

Directed Cellular Self-Assembly to Fabricate Cell-Derived Tissue Rings for Biomechanical Analysis and Tissue Engineering

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do not have suitable autologous donor vessels due to disease progression or previous harvest. The aim of vascular tissue engineering is to develop a suitable alternative source for these bypass grafts. In addition, engineered vascular tissue may prove valuable as living vascular models to study cardiovascular diseases. Several promising approaches to engineering blood vessels have been explored, with many recent studies focusing on development and analysis of cell-based methods.2-5 Herein, we present a method to rapidly self-assemble cells into 3D tissue rings that can be used in vitro to model vascular tissues.
To do this, suspensions of smooth muscle cells are seeded into round-bottomed annular agarose wells. The non-adhesive properties of the agarose allow the cells to settle, aggregate and contract around a post at the center of the well to form a cohesive tissue ring.6,7 These rings can be cultured for several days prior to harvesting for mechanical, physiological, biochemical, or histological analysis. We have shown that these cell-derived tissue rings yield at 100-500 kPa ultimate tensile strength8 which exceeds the value reported for other tissue engineered vascular constructs cultured for similar durations (&lt;30 kPa).9,10 Our results demonstrate that robust cell-derived vascular tissue ring generation can be achieved within a short time period, and offers the opportunity for direct and quantitative assessment of the contributions of cells and cell-derived matrix (CDM) to vascular tissue structure and function.

This article outlines a versatile method to create cell-derived tissue rings by cellular self-assembly. Smooth muscle cells seeded into ring-shaped agarose wells aggregate and contract to form robust three-dimensional (3D) tissues within 7 days. Millimeter-scale tissue rings are conducive to mechanical testing and serve as building blocks for tissue assembly.

Dirigida celular de auto-ensamblaje para fabricar células derivadas de tejido para el análisis de anillos de Biomecánica e Ingeniería de Tejidos

Each year, hundreds of thousands of patients undergo coronary artery bypass surgery in the United States.1 Approximately one third of these patients do ...

Investigación

Bioingeniería

Non-contact, Label-free Monitoring of Cells and Extracellular Matrix using Raman Spectroscopy

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, currently available routine methods require processing steps and alter sample integrity. Raman spectroscopy is a fast method that enables the measurement of biological samples without the need for further processing steps. This laser-based technology detects the inelastic scattering of monochromatic light.6 As every chemical vibration is assigned to a specific Raman band (wavenumber in cm-1), each biological sample features a typical spectral pattern due to their inherent biochemical composition.7-9 Within Raman spectra, the peak intensities correlate with the amount of the present molecular bonds.1 Similarities and differences of the spectral data sets can be detected by employing a multivariate analysis (e.g. principal component analysis (PCA)).10
Here, we perform Raman spectroscopy of living cells and native tissues. Cells are either seeded on glass bottom dishes or kept in suspension under normal cell culture conditions (37 &deg;C, 5% CO2) before measurement. Native tissues are dissected and stored in phosphate buffered saline (PBS) at 4 &deg;C prior measurements. Depending on our experimental set up, we then either focused on the cell nucleus or extracellular matrix (ECM) proteins such as elastin and collagen. For all studies, a minimum of 30 cells or 30 random points of interest within the ECM are measured. Data processing steps included background subtraction and normalization.

Raman spectroscopy is a suitable technique for the non-contact, label-free analysis of living cells, tissue-engineered constructs and native tissues. Source-specific spectral fingerprints can be generated and analyzed using multivariate analysis.

Sin contacto, libre de marca Seguimiento de células y matriz extracelular utilizando espectroscopia Raman

Non-destructive, non-contact and label-free technologies to monitor cell and tissue cultures are needed in the field of biomedical research.1-5 However, ...

Engineering Skeletal Muscle Tissues from Murine Myoblast Progenitor Cells and Application of Electrical Stimulation

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to study pressure ulcers, for regenerative medicine and as a meat alternative 1. The first reported 3D muscle constructs have been made many years ago and pioneers in the field are Vandenburgh and colleagues 2,3. Advances made in muscle tissue engineering are not only the result from the vast gain in knowledge of biochemical factors, stem cells and progenitor cells, but are in particular based on insights gained by researchers that physical factors play essential roles in the control of cell behavior and tissue development. State-of-the-art engineered muscle constructs currently consist of cell-populated hydrogel constructs. In our lab these generally consist of murine myoblast progenitor cells, isolated from murine hind limb muscles or a murine myoblast cell line C2C12, mixed with a mixture of collagen/Matrigel and plated between two anchoring points, mimicking the muscle ligaments. Other cells may be considered as well, e.g. alternative cell lines such as L6 rat myoblasts 4, neonatal muscle derived progenitor cells 5, cells derived from adult muscle tissues from other species such as human 6 or even induced pluripotent stem cells (iPS cells) 7. Cell contractility causes alignment of the cells along the long axis of the construct 8,9 and differentiation of the muscle progenitor cells after approximately one week of culture. Moreover, the application of electrical stimulation can enhance the process of differentiation to some extent 8. Because of its limited size (8 x 2 x 0.5 mm) the complete tissue can be analyzed using confocal microscopy to monitor e.g. viability, differentiation and cell alignment. Depending on the specific application the requirements for the engineered muscle tissue will vary; e.g. use for regenerative medicine requires the up scaling of tissue size and vascularization, while to serve as a meat alternative translation to other species is necessary.

Engineered muscle tissue has great potential in regenerative medicine, as disease model and also as an alternative source for meat. Here we describe the engineering of a muscle construct, in this case from mouse myoblast progenitor cells, and the stimulation by electrical pulses.

Los tejidos de ingeniería músculo esquelético de células mioblastos murinos progenitoras y aplicación de estimulación eléctrica

Engineered muscle tissues can be used for several different purposes, which include the production of tissues for use as a disease model in vitro, e.g. to ...

Tissue Engineering of Tumor Stromal Microenvironment with Application to Cancer Cell Invasion

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and invasion. Rat tail type I collagen and/or matrix derived from Engelbreth-Holm-Swarm mouse sarcoma cells have been traditionally employed as the substrates to model the matrix or stromal microenvironment into which mesenchymal cells (usually fibroblasts) are populated. Although experiments using such matrices are very informative, it can be argued that due to an overriding presence of a single protein (such as in type I Collagen) or a high content of basement membrane components and growth factors (such as in matrix derived from mouse sarcoma cells), these substrates do not best reflect the contribution to matrix composition made by the stromal cells themselves. To study native matrices produced by primary dermal fibroblasts isolated from patients with a tumor prone, genetic blistering disorder (recessive dystrophic epidermolysis bullosa), we have adapted an existing native matrix protocol to study tumor cell invasion. Fibroblasts are induced to produce their own matrix over a prolonged period in culture. This native matrix is then detached from the culture dish and epithelial cells are seeded onto it before the entire coculture is raised to the air-liquid interface. Cellular differentiation and/or invasion can then be assessed over time. This technique provides the ability to assess epithelial-mesenchymal cell interactions in a 3D setting without the need for a synthetic or foreign matrix with the only disadvantage being the prolonged period of time required to produce the native matrix. Here we describe the application of this technique to assess the ability of a single molecule expressed by fibroblasts, type VII collagen, to inhibit tumor cell invasion.

Tissue engineered fibroblast-derived native matrix is an emerging tool to generate a stromal substrate which supports epithelial cell proliferation and differentiation. Here a protocol applying this methodology to assess the impact of different stromal cell types on tumor cell biology is presented.

Ingeniería de tejidos de tumor del estroma Microambiente con Aplicación a la invasión de células cancerosas

3D organotypic cultures of epithelial cells on a matrix embedded with mesenchymal cells are widely used to study epithelial cell differentiation and ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Ingeniería 3D Cellularized colágeno Geles para Vascular Regeneración Tisular

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Microentornos-Sandwich como para aprovechar Celulares / Materiales Interacciones

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Treatment of Ligament Constructs with Exercise-conditioned Serum: A Translational Tissue Engineering Model

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, and translation of findings is often poor. Thus, there is ample opportunity for alternative experimental approaches. Here we present an approach in which human cells are isolated from human anterior cruciate ligament tissue remnants, expanded in culture, and used to form engineered ligaments. Exercise alters the biochemical milieu in the blood such that the function of many tissues, organs and bodily processes are improved. In this experiment, ligament construct culture media was supplemented with experimental human serum that has been &#39;conditioned&#39; by exercise. Thus the intervention is more biologically relevant since an experimental tissue is exposed to the full endogenous biochemical milieu, including binding proteins and adjunct compounds that may be altered in tandem with the activity of an unknown agent of interest. After treatment, engineered ligaments can be analyzed for mechanical function, collagen content, morphology, and cellular biochemistry. Overall, there are four major advantages versus traditional monolayer culture and animal models, of the physiological model of ligament tissue that is presented here. First, ligament constructs are three-dimensional, allowing for mechanical properties (i.e., function) such as ultimate tensile stress, maximal tensile load, and modulus, to be quantified. Second, the enthesis, the interface between boney and sinew elements, can be examined in detail and within functional context. Third, preparing media with post-exercise serum allows for the effects of the exercise-induced biochemical milieu, which is responsible for the wide range of health benefits of exercise, to be investigated in an unbiased manner. Finally, this experimental model advances scientific research in a humane and ethical manner by replacing the use of animals, a core mandate of the National Institutes of Health, the Center for Disease Control, and the Food and Drug Administration.

We present a model of ligament tissue in which three-dimensional constructs are treated with the human exercise-conditioned serum and analyzed for collagen content, function, and cellular biochemistry.

Tratamiento de las estructuras de ligamentos con suero acondicionado con ejercicio: Un modelo de ingeniería de tejidos traslacional

In vitro experiments are essential to understand biological mechanisms; however, the gap between monolayer tissue culture and human physiology is large, ...

Recombinant Collagen I Peptide Microcarriers for Cell Expansion and Their Potential Use As Cell Delivery System in a Bioreactor Model

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation purposes. The process to generate such tissues is complex, and includes an appropriate combination of specific cell types, scaffolds, and physical or biochemical stimuli to guide cell growth and differentiation. Microcarriers represent an appealing tool to expand cells in a three-dimensional (3D) microenvironment, since they provide higher surface-to volume ratios and mimic more closely the in vivo situation compared to traditional two-dimensional methods. The vascular system, supplying oxygen and nutrients to the cells and ensuring waste removal, constitutes an important building block when generating engineered tissues. In fact, most constructs fail after being implanted due to lacking vascular support. In this study, we present a protocol for endothelial cell expansion on recombinant collagen-based microcarriers under dynamic conditions in spinner flask and bioreactors, and we explain how to determine in this setting cell viability and functionality. In addition, we propose a method for cell delivery for vascularization purposes without additional detachment steps necessary. Furthermore, we provide a strategy to evaluate the cell vascularization potential in a perfusion bioreactor on a decellularized&#160;biological matrix. We believe that the use of the presented methods could lead to the development of new cell-based therapies for a large range of tissue engineering applications in the clinical practice.

We propose a cell expansion protocol on macroporous microcarriers and their use as delivery system in a perfusion bioreactor to seed a decellularized tissue matrix. We also include different techniques to determine cell proliferation and viability of cells cultured on microcarriers. Furthermore, we demonstrate functionality of cells after bioreactor cultures.

Recombinante colágeno péptido Microcarriers para la extensión celular y su posible uso como sistema de células en un biorreactor modelo

Tissue engineering is a promising field, focused on developing solutions for the increasing demand on tissues and organs regarding transplantation ...

A Novel Tenorrhaphy Suture Technique with Tissue Engineered Collagen Graft to Repair Large Tendon Defects

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily identified and used. Currently, this gap is filled with tendon auto-, allo-, xeno-, or artificial grafts, but clinical methods to secure them are not necessarily translatable to animals because of the scale. In order to evaluate new biomaterials or study a tendon graft made up of collagen type 1, we have developed a modified suture technique to help maintain the engineered tendon in alignment with the tendon ends. Mechanical properties of these grafts are inferior to the native tendon. To incorporate engineered tendon into clinically relevant models of loaded repair, a strategy was adopted to offload the tissue engineered tendon graft and allow for the maturation and integration of the engineered tendon in vivo until a mechanically sound neo-tendon was formed. We describe this technique using incorporation of the collagen type 1 tissue engineered tendon construct.

In this paper, we present an in vitro and in situ protocol to repair a tendon gap of up to 1.5 cm by filling it with engineered collagen graft. This was performed by developing a modified suture technique to take the mechanical load until the graft matures into the host tissue.

Una nueva técnica de sutura de tenorrafia con injerto de colágeno de ingeniería tisular para reparar defectos grandes del tendón

Surgical management of large tendon defects with tendon grafts is challenging, as there are a finite number of sites where donors can be readily ...

Pancreatic Tissue-Derived Extracellular Matrix Bioink for Printing 3D Cell-Laden Pancreatic Tissue Constructs

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary complications. However, islet transplantation still has several limitations such as the low viability of transplanted islets due to poor islet engraftment and hostile environments. In addition, the insulin-producing cells differentiated from human pluripotent stem cells have limited ability to secrete sufficient hormones that can regulate the blood glucose level; therefore, improving the maturation by culturing cells with proper microenvironmental cues is strongly required. In this article, we elucidate protocols for preparing a pancreatic tissue-derived decellularized extracellular matrix (pdECM) bioink to provide a beneficial microenvironment that can increase glucose sensitivity of pancreatic islets, followed by describing the processes for generating 3D pancreatic tissue constructs using a microextrusion-based bioprinting technique.

Decellularized extracellular matrix (dECM) can provide suitable microenvironmental cues to recapitulate the inherent functions of target tissues in an engineered construct. This article elucidates the protocols for the decellularization of pancreatic tissue, evaluation of pancreatic tissue-derived dECM bioink, and generation of 3D pancreatic tissue constructs using a bioprinting technique.

Bioink de matriz extracelular derivada del tejido pancreático para imprimir construcciones de tejido pancreático laden de células 3D

The transplantation of pancreatic islets is a promising treatment for patients who suffer from type 1 diabetes accompanied by hypoglycemia and secondary ...