Agradecemos a George Giudice y Steven Eliason por donar fibroblastos dérmicos humanos y a Ramesh Raghupathy por su ayuda con el algoritmo de seguimiento de cepas. El apoyo para este trabajo fue proporcionado por una beca de asistencia para graduados del Departamento de Educación de los Estados Unidos en áreas de necesidad nacional (GAANN P200A120071).

1. Preparación de la plantilla
Prepare una plantilla en Parafilm para diseñar la ubicación de cada explant, siguiendo la geometría deseada (Figuras 1A y 1B). Espaciar cada explanta aproximadamente 1-2 mm de distancia. Esta distancia corresponde a un espaciamiento ideal para generar alineación de fibras entre explantes. Coloque la plantilla debajo del área del vidrio de cubierta donde se preparará la muestra con cinta adhesiva.
2. Esterilización
Limpie a fondo todos los componentes del biorreactor usando etanol al 70% y esterilice durante 2-3 horas bajo luz UV antes de la experimentación. Si se está utilizando un recipiente alternativo en lugar de un biorreactor, se deben utilizar técnicas de esterilización adecuadas. Consulte el paso 4.12 para obtener comentarios sobre el uso de una placa de Petri con fondo de vidrio.
3. Preparación de gel de fibrina
Después de que los componentes del biorreactor hayan sido esterilizados, eche una capa delgada de gel de fibrina en la superficie del coverglass. Los detalles para preparar las soluciones del stock del fibrinógeno y de la trombina para hacer los geles de la fibrina de 6,6 mg/ml se describen en lijadora y otros. 13 Un protocolo similar por Ye et al. 19 para hacer geles de fibrina de 3,3 mg/ml también se puede ver en el sitio web de JoVE.
<ol><li>Prepare una solución de microperlas fluorescentes en DMEM a una concentración de 10 millones de perlas/ml. Las cuentas se utilizarán para ayudar a rastrear los desplazamientos de gel. Para lograr esta concentración, combine 0,017 ml de solución de microperla y 0,149 ml de DMEM en un tubo de microcentrífuga.</li>
	<li>Sonicar esta suspensión durante 10 min para dispersar las cuentas y homogeneizar la solución.</li>
	<li>Solución de fibrina — En un tubo c de 15 ml, mezcle 0.22 ml de solución común de fibrinógeno con 0.44 ml de tampón HEPES de 20 mM. Añadir los 0,1667 ml de DMEM con microperlas creadas en el paso 3.1.</li>
	<li>Solución de trombina — En un tubo c separado de 15 ml, mezcle juntos 0.0328 ml de solución común de trombina, 0.131 ml de tampón HEPES de 20 mM, y 0.00246 ml de 2 M CaCl2.</li>
	<li>Mezcle cuidadosamente la solución de trombina (paso 3.4) con la solución de fibrinógeno (paso 3.3) pipeteando hacia arriba y hacia abajo 5-10x hasta que la solución se distribuya uniformemente. Evite introducir burbujas tanto como sea posible. Para reducir la cantidad de burbujas producidas, tenga cuidado de no descargar completamente la pipeta mientras se mezcla.</li>
	<li>La adición de trombina hará que la solución se gelifique rápidamente (~ 30 seg). Pipetear la solución mixta en la cubierta de vidrio tan pronto como sea posible. Deje que el gel se polimerice en RT.</li>
	<li>Selle el biorreactor, inserte los bloques de calentamiento y conecte los termopares al controlador de temperatura. Incubar el gel a 37 °C durante 15-30 min.</li>
</ol>4. Preparación de explantes celulares
<ol><li>Retire el medio del matraz T-75 que contiene las células de fibroblastos dérmicos humanos.</li>
	<li>Enjuague cuidadosamente la superficie con aproximadamente 5 ml de solución salina tamponada con fosfato (PBS) para eliminar las proteínas séricas. Añadir 1 ml de tripsina-EDTA e incubar durante 3 min, o hasta que las células se hayan levantado.</li>
	<li>Después de que las células se han levantado, gire la suspensión hacia abajo en una centrífuga a 200 x g durante 5 min. Retire el sobrenadante y resuspiente el pellet en un volumen de DMEM que permitirá una concentración final de 20 millones de células/ml.</li>
	<li>Mientras las células están girando hacia abajo en la centrífuga desconecte el biorreactor de los bloques de calefacción y los termopares. Transfiera el biorreactor a un gabinete de bioseguridad y retire cuidadosamente la tapa siguiendo técnicas asceptivas.</li>
	<li>Cree explantes pipeteando 0,3 μl de la suspensión celular sobre el gel de fibrina polimerizada, siguiendo el patrón de la plantilla. Cada explant debe contener aproximadamente 6.000 células. Asegúrese de que se utilizan puntas de micropipeta de bajo volumen (0.1-10 μl).</li>
	<li>Deje que las células se asienten y se adhieran a la matriz de fibrina durante 1 hora a 37 °C.</li>
	<li>Con el biorreactor todavía abierto, agregue aproximadamente 5 ml de DMEM suplementado con suero bovino fetal del 10% (FBS), penicilina-estreptomicina del 1%, anfotericina B del 0,1%, y aprotinin de 10 mg/ml directamente en el compartimiento del biorreactor. DMEM es bicarbonato tamponado y requiere 5% de CO2 para mantener un pH neutro. Dado que el biorreactor no se suministra con CO2,acondicione el medio en una incubadora con 5% deCO2 durante 2-3 horas antes de su uso. La aprotinina es un inhibidor de la serina proteasa que es ampliamente utilizado para reducir la tasa de degradación de la fibrina20.</li>
	<li>Vuelva a seseer el biorreactor. Use una jeringa para administrar 5 ml adicionales de co2 acondicionado medio a través del accesorio de púas en el puerto de entrada. Dispense el medio lentamente y asegúrese de que todo el volumen de la cámara del biorreactor esté lleno. Retire cuidadosamente las burbujas que se forman en el biorreactor.</li>
	<li>Suministre un medio fresco acondicionado de CO2 al 5% al biorreactor durante todo el experimento para mantener el pH, suministrar nutrientes y eliminar los productos de desecho. Configure una bomba de jeringa con una jeringa de 10 ml o 30 ml llena de medio acondicionado de CO2 al 5%. Conecte la jeringa directamente al puerto de entrada en la tapa del biorreactor con el tubo estéril instalado en Luer-lock. Retire el accesorio macho y coloque el tubo en el accesorio de púas del biorreactor (consulte la Figura 1C).</li>
	<li>Establezca la tasa de perfusión en 0,01 ml/min. Conecte las piezas modificadas de tubería a ambos puertos de salida y coloque los extremos en un beaker de 100 ml para recoger los residuos.</li>
	<li>Utilice un conector de laboratorio para establecer las alturas de las alimentaciones de entrada y salida para que no se desarrolle un diferencial de presión en el biorreactor (consulte la Figura 1D para referencia).</li>
	<li>Si un biorreactor no está disponible, prepare muestras en placas de Petri con fondo de vidrio de 35 mm con tapas de vidrio. Utilice uno con un tamaño de cubrebocas que esté optimizado para el conjunto específico de objetivos que se utilizarán. Las muestras que se preparan en placas de Petri deben mantenerse en una incubadora a 37 °C y al 5% de CO2. Nota: El poliestireno despolariza la luz e interferirá con las imágenes DIC, por lo que se deben usar tapas de vidrio si se realizarán imágenes DIC. El contraste de fase es una modalidad alternativa conveniente de la proyección de imagen. La transferencia de platos entre la incubadora y el microscopio dificultará el registro de imágenes. Si las imágenes no se registran correctamente, la cepa calculada en la Sección 6 no será exacta.</li>
</ol>5. Imágenes de lapso de tiempo
<ol><li>Una vez que la muestra ha sido preparada, vuelva a sesecar el biorreactor, vuelva a conectar los bloques de calentamiento y los termopares, y ajuste la temperatura del biorreactor a 37 °C.</li>
	<li>Fije el biorreactor en la etapa motorizada montada en el microscopio. Coloque el objetivo DIC 20X bajo el puerto de vista. Un objetivo de aumento más bajo es aceptable, particularmente si una etapa motorizada de precisión no está disponible. Asegúrese de que el polarizador, el analizador y el prisma estén en su lugar. Alternativamente, las muestras también se pueden fotoizar utilizando el contraste de fase.</li>
	<li>Abra el software de imágenes.</li>
	<li>Centrar el objetivo en el área entre los tres explantes.</li>
	<li>Guarde las coordenadas (x, y y z) de esta ubicación en el software de creación de imágenes.</li>
	<li>Con el fin de obtener imágenes de toda el área entre y alrededor de los explantes, seleccione la opción de adquirir una imagen grande y especifique el tamaño del área. Esto permitirá la adquisición de varias imágenes alrededor del área especificada y creará una imagen en mosaico que representa una región más grande de la muestra.</li>
	<li>Establezca el tiempo de exposición y la intensidad de la luz en los valores más bajos posibles para evitar la muerte celular causada por la fototoxicidad, al tiempo que proporciona una resolución suficiente para discriminar entre células, microperlas y fibras de fibrina.</li>
</ol>6. Seguimiento de cepas
Para obtener más información e instrucciones sobre el software de seguimiento de cepas (Figura 2) utilizado, consulte Raghupathy et al. 21 El algoritmo es un código MATLAB personalizado que se puede descargar desde <a href="http://www.license.umn.edu/default.aspx">http://www.license.umn.edu/default.aspx</a>. Tenga en cuenta que las imágenes DIC a menudo tienen suficiente textura para el seguimiento de la deformación. Las microperlas se incluyen para servir como un control en los campos de deformación calculados. Si se va a realizar un seguimiento de la deformación, es fundamental que las imágenes obtenidas se tomen exactamente en el mismo lugar para que las imágenes se registren. Las imágenes no registradas producirán cepas espurias.

<ol>
	<li>Grinnell, F., Petroll WM, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+motility+and+mechanics+in+three-dimensional+collagen+matrices.">Cell motility and mechanics in three-dimensional collagen matrices.</a> Annu. Rev. Cell. Dev. Biol. 26, 335-361 (2001).</li><li>Sander, E. A., 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=.">.</a> Biomimetic collagen tissues: collagenous tissue engineering and other applications. In: Collagen: Structure and Mechanics. , (2008).</li><li>Pedersen JA, ., Swartz MA, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanobiology+in+the+third+dimension.">Mechanobiology in the third dimension.</a> Ann. Biomed. Eng. 33 (11), 1469-1490 (2005).</li><li>Bell, E., Ivarsson, B., Merrill, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+a+tissue-like+structure+by+contraction+of+collagen+lattices+by+human+fibroblasts+of+different+proliferative+potential+in+vitro.">Production of a tissue-like structure by contraction of collagen lattices by human fibroblasts of different proliferative potential in vitro.</a> Proc. Natl. Acad. Sci. U.S.A. 76 (3), 1274 (1979).</li><li>Guidry, C., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+the+mechanism+of+hydrated+collagen+gel+reorganization+by+human+skin+fibroblasts.">Studies on the mechanism of hydrated collagen gel reorganization by human skin fibroblasts.</a> J. Cell. Sci. 79 (1), 67-81 (1985).</li><li>Fluck, J., Querfeld, C., Cremer, A., Niland, S., Krieg, T., Sollberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+human+primary+fibroblasts+undergo+apoptosis+in+three-dimensional+contractile+collagen+gels.">Normal human primary fibroblasts undergo apoptosis in three-dimensional contractile collagen gels.</a> J. Invest. Dermatol. 110 (2), 153-157 (1998).</li><li>Rosenfeldt, H., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+quiescence+and+the+disruption+of+ERK+signaling+in+mechanically+unloaded+collagen+matrices.">Fibroblast quiescence and the disruption of ERK signaling in mechanically unloaded collagen matrices.</a> J. Biol. Chem. 275 (5), 3088-3092 (2000).</li><li>Nakagawa, S., Pawelek, P., Grinnell, F. <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+culture+of+fibroblasts+in+contracted+collagen+gels:+Effects+on+cell+growth+and+biosynthetic+activity.">Long-term culture of fibroblasts in contracted collagen gels: Effects on cell growth and biosynthetic activity.</a> J. Invest. Dermatol. 93 (6), 792-798 (1989).</li><li>Harris, A. K., Stopak, D., Wild, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+traction+as+a+mechanism+for+collagen+morphogenesis.">Fibroblast traction as a mechanism for collagen morphogenesis.</a> Nature. 290, 249-251 (1981).</li><li>Stopak, D., Harris AK, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Connective+tissue+morphogenesis+by+fibroblast+traction:+I.+tissue+culture+observations.">Connective tissue morphogenesis by fibroblast traction: I. tissue culture observations.</a> Dev. Biol. 90 (2), 383-398 (1982).</li><li>Sawhney, R. K., Howard, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Slow+local+movements+of+collagen+fibers+by+fibroblasts+drive+the+rapid+global+self-organization+of+collagen+gels.">Slow local movements of collagen fibers by fibroblasts drive the rapid global self-organization of collagen gels.</a> J. Cell. Biol. 157 (6), 1083-1092 (2002).</li><li>Provenzano, P. P., Inman, D. R., Eliceiri, K. W., Trier, S. M., Keely, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact+guidance+mediated+three-dimensional+cell+migration+is+regulated+by+rho/ROCK-dependent+matrix+reorganization.">Contact guidance mediated three-dimensional cell migration is regulated by rho/ROCK-dependent matrix reorganization.</a> Biophys. J. 95 (11), 5374 (2008).</li><li>Sander, E., Barocas, V., Tranquillo, R. <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 (2), 714-729 (2011).</li><li>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=An+anisotropic+biphasic+theory+of+tissue-equivalent+mechanics:+the+interplay+among+cell+traction,+fibrillar+network+deformation,+fibril+alignment,+and+cell+contact+guidance.">An anisotropic biphasic theory of tissue-equivalent mechanics: the interplay among cell traction, fibrillar network deformation, fibril alignment, and cell contact guidance.</a> J. Biomech. Eng. 119, 137-146 (1997).</li><li>Neidert, M. R., 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=Tissue-engineered+valves+with+commissural+alignment.">Tissue-engineered valves with commissural alignment.</a> Tissue Eng. 12 (4), 891-903 (2006).</li><li>Robinson PS, ., Robinson PS, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Planar+biaxial+behavior+of+fibrin-based+tissue-engineered+heart+valve+leaflets.">Planar biaxial behavior of fibrin-based tissue-engineered heart valve leaflets.</a> Tissue Eng. Part A. 15 (10), 2763-2772 (2009).</li><li>Grassl, E., Oegema, T., Tranquillo, R. <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+an+alternative+biopolymer+to+type+I+collagen+for+the+fabrication+of+a+media+equivalent.">Fibrin as an alternative biopolymer to type I collagen for the fabrication of a media equivalent.</a> J. Biomed. Mater. Res. 60 (4), 607-612 (2002).</li><li>Paten, J. A., Zareian, R., Saeidi, N., Melotti, S. A., Ruberti, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Design+and+performance+of+an+optically+accessible,+low-volume,+mechanobioreactor+for+long-term+study+of+living+constructs.">Design and performance of an optically accessible, low-volume, mechanobioreactor for long-term study of living constructs.</a> Tissue Eng. Part C Methods. 17 (7), 775-788 (2001).</li><li>Ye KY, ., Sulli van KE, ., Black LD, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Encapsulation+of+cardiomyocytes+in+a+fibrin+hydrogel+for+cardiac+tissue+engineering.">Encapsulation of cardiomyocytes in a fibrin hydrogel for cardiac tissue engineering.</a> J. Vis. Exp. (55), (2011).</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 (10), 3261-3270 (2010).</li><li>Raghupathy, R., Witzenburg, C., Lake, S. P., Sander, E. A., 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=Identification+of+regional+mechanical+anisotropy+in+soft+tissue+analogs.">Identification of regional mechanical anisotropy in soft tissue analogs.</a> J. Biomech. Eng. 133, (2011).</li><li>Robinson, P. S., Johnson, S. L., Evans, M. C., 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=Functional+tissue-engineered+valves+from+cell-remodeled+fibrin+with+commissural+alignment+of+cell-produced+collagen.">Functional tissue-engineered valves from cell-remodeled fibrin with commissural alignment of cell-produced collagen.</a> Tissue Eng. Part A. 14 (1), 83-95 (2008).</li><li>Gabbiani, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+myofibroblast+in+wound+healing+and+fibrocontractive+diseases.">The myofibroblast in wound healing and fibrocontractive diseases.</a> J. Pathol. 200 (4), 500-503 (2003).</li><li>PaszeK, M. 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=Tensional+homeostasis+and+the+malignant+phenotype.">Tensional homeostasis and the malignant phenotype.</a> Cancer Cell. 8 (3), 241-254 (2005).</li><li>Grinnell, F., Rocha L, B., Iucu, C., Rhee, S., Jiang, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nested+collagen+matrices:+A+new+model+to+study+migration+of+human+fibroblast+populations+in+three+dimensions.">Nested collagen matrices: A new model to study migration of human fibroblast populations in three dimensions.</a> Exp. Cell. Res. 312 (1), 86-94 (2006).</li><li>Miron-Mendoza, M., Seemann, J., Grinnell, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+differential+regulation+of+cell+motile+activity+through+matrix+stiffness+and+porosity+in+three+dimensional+collagen+matrices.">The differential regulation of cell motile activity through matrix stiffness and porosity in three dimensional collagen matrices.</a> Biomaterials. 31 (25), 6425-6435 (2010).</li><li>Wakatsuki, T., Kolodney MS, ., Zahalak GI, ., Elson EL, . <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+mechanics+studied+by+a+reconstituted+model+tissue.">Cell mechanics studied by a reconstituted model tissue.</a> Biophys. J. 79 (5), 2353-2368 (2000).</li><li>De Jesus, A., Aghvami, M., Sander, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast-mediated+fiber+realignment+in+fibrin+gels.">Fibroblast-mediated fiber realignment in fibrin gels.</a> , (2013).</li><li>Aghvami, M., Barocas, V. H., Sander, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiscale+mechanical+simulations+of+cell+compacted+collagen+gels.">Multiscale mechanical simulations of cell compacted collagen gels.</a> J. Biomech. Eng. 135, (2013).</li></ol>

No se declaran conflictos de intereses.

Este protocolo fue desarrollado con el propósito de observar y cuantificar la mecánica involucrada en la remodelación de ECM mediada por células. Tales procesos son la base de una serie de fenómenos biológicos y tienen implicaciones importantes para la ingeniería de tejidos2,22,la reducción de la cicatriz1,23,y la comprensión de la remodelación patológica de los tejidos12,24. El uso de la microscopia DIC del lapso de tiempo permite que uno resuelva y cuantifique la dislocación...

Una herramienta importante para el estudio de las interacciones célula-matriz es el gel de colágeno poblado por células1,2. El gel proporciona un entorno 3D que está más cerca del carácter in vivo del tejido y es más adecuado para comprender el comportamiento celular que el que ofrecen los cultivos 2D tradicionales3. Los primeros estudios en los que los fibroblastos se distribuían homogéneamente dentro de un gel de colágeno encontraron que las células consolidan rápidamente las fibras de colágeno y compactan el gel4,5. Los fibroblastos contráctiles en geles flotantes libres pasan a un estado quieto poco después de que el gel haya alcanzado completamente la compactación1,6,7. Los fibroblastos en geles que están restringidos en los límites permanecen en un estado activo y sintético8 y generan alineación de fibras de una manera dependiente de la geometría del gel y las restricciones externas5,9. Las diferencias en la actividad celular parecen ser el resultado de la tensión interna (o la falta de ella) que se desarrolla a medida que las células ejercen fuerzas de tracción a través de integrinas sobre las fibras de colágeno en el gel.
Una variante de esta técnica consiste en colocar explantes de fibroblastos(es decir, grupos de células) a una distancia de un gel de colágeno y observar las interacciones célula-matriz y el desarrollo gradual de la alineación de la fibra entre los explantes (a veces llamados correas similares a ligamentos)10-12. La principal ventaja del sistema explant es que permite organizar las células en patrones geométricos simples, lo que facilita la visualización y sonda de los mecanismos subyacentes a la realineación de fibras impulsadas por células. Estos patrones de alineación , que dependen principalmente de la interacción entre las fuerzas de tracción celular, la distribución espacial celular, la geometría del gel y las restricciones mecánicas en el gel , son importantes de entender porque desempeñan un papel central en la organización global del tejido, la función mecánica y el entorno mecánico local13.
En el campo de la ingeniería de tejidos, una estrategia para producir tejidos mecánicamente funcionales y de ingeniería implica controlar el patrón de alineación de fibras que se desarrolla a partir de la compactación celular para que el tejido diseñado posea una alineación de fibras que imite la del tejido nativo14,15. Tal alineación se cree necesaria para que los tejidos diseñados repliquen el comportamiento mecánico complejo de los tejidos nativos. Una modificación de esta estrategia es sustituir el gel de colágeno por un gel de fibrina16. El gel de fibrina desarrolla un patrón de alineación similar al de un gel de colágeno durante la compactación. Con el tiempo, la fibrina se degrada y se reemplaza con ECM sintetizado por células que sigue el patrón inicial de alineación de la fibra de fibrina. La construcción de ingeniería resultante ha mejorado significativamente las propiedades mecánicas en comparación con las construcciones derivadas del gel de colágeno17.
El proceso de alineación y los eventos de remodelación posteriores en geles de fibrina proceden de una manera complicada y poco entendida. Para caracterizar mejor estas interacciones y su efecto sobre el comportamiento celular y la remodelación de ecm, hemos desarrollado un procedimiento que se basa en el método explant. En este método, los explantes de fibroblastos se colocan en un gel de fibrina en diferentes patrones geométricos. Los geles se mantienen en un biorreactor18controlado ambientalmente y montado en un microscopio, y el proceso de compactación y realineación de la fibra se supervisa con microscopía de contraste de interferencia diferencial (DIC) de lapso de tiempo. Los campos de desplazamiento se cuantifican con algoritmos personalizados. Los datos obtenidos de estos experimentos tienen implicaciones de amplia gama para una serie de procesos, incluyendo la optimización de las estrategias de ingeniería de tejidos, la mejora de la cicatrización de heridas y el tratamiento de la remodelación patológica del tejido.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Sigma-Aldrich</td>
			<td>F8630</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>T4648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>11965-092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>15140-122</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A2942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>H0887</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>223506</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Gibco</td>
			<td>25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Invitrogen</td>
			<td>3000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lonza</td>
			<td>DE14-701F</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Molecular Probes</td>
			<td>F8858</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>A10483-01</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">GIBCO</td>
			<td>11430-030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fisher-Scientific</td>
			<td>SS264-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sigma-Aldrich</td>
			<td>A3428-25MG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:297px;">Biotense Bioreactor</td>
			<td style="width:85px;">ADMET</td>
			<td style="width:125px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ti-Eclipe Microscope</td>
			<td>Nikon</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Bottom Petri Dish</td>
			<td>MatTek</td>
			<td>P35G-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"># 0 35 mm Glass Top Petri Dish</td>
			<td>MatTek</td>
			<td>P35GTOP-0-20-C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Luer fittings, PVC tubing with Luer ends</td>
			<td>Cole-Parmer</td>
			<td>30600-65</td>
		</tr>
	</tbody>
</table>

observing and quantifying fibroblast-mediated fibrin gel compaction

Las células incrustadas en los geles de colágeno y fibrina se unen y ejercen fuerzas de tracción sobre las fibras del gel. Estas fuerzas pueden conducir a la reorganización local y global y la realineación de la microestructura de gel. Este proceso procede de una manera compleja que depende en parte de la interacción entre la ubicación de las células, la geometría del gel y las restricciones mecánicas en el gel. Para comprender mejor cómo estas variables producen patrones globales de alineación de fibras, utilizamos microscopía de contraste de interferencia diferencial de lapso de tiempo (DIC) junto con un biorreactor controlado ambientalmente para observar el proceso de compactación entre explantes geométricamente espaciados (grupos de fibroblastos). A continuación, las imágenes se analizan con un algoritmo de procesamiento de imágenes personalizado para obtener mapas de la cepa. La información obtenida de esta técnica se puede utilizar para sondear la mecanobiología de varias interacciones de la célula-matriz, que tiene implicaciones importantes para entender procesos en la herida que cura, el desarrollo de la enfermedad, y los usos de la ingeniería del tejido.

La remodelación de tejidos es un proceso complejo que es impulsado en parte por interacciones físicas recíprocas entre las células y la matriz circundante. Las células reorganizan las fibras circundantes y generan tensión en la red de fibra. La alineación de las fibras y el entorno mecánico a su vez controla el comportamiento celular, de modo que tanto las células como la matriz se reorganizan globalmente para producir tejido remodelado. En este experimento, las células de los explantes, inicialmente redondas e...

Watch this Scientific Journal Video about Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction at JoVE.com

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

La microscopia del lapso de tiempo y las técnicas de proceso de imagen fueron utilizadas para observar y para analizar la compactación fibroblasto-mediada del gel y el realineamiento de la fibra de la fibrina en un biorreactor ambientalmente controlado durante un período de 48 horas.

Observación Y Cuantificación De Fibroblastos Mediada Por Fibrina Gel Compactamiento

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

observing-and-quantifying-fibroblast-mediated-fibrin-gel-compaction

Research

JoVE Journal

Bioengineering

5.9K vistas.  University of Iowa. La microscopia del lapso de tiempo y las técnicas de proceso de imagen fueron utilizadas para observar y para analizar la compactación fibroblasto-mediada del gel y el realineamiento de la fibra de la fibrina en un biorreactor ambientalmente controlado durante un período de 48 horas.

Video: Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Preparation of Complaint Matrices for Quantifying Cellular Contraction

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling. Focal adhesions are macromolecular assemblies that couple the contractile F-actin cytoskeleton to the ECM. This connection allows for the transmission of intracellular mechanical forces across the cell membrane to the underlying substrate. Recent work has shown the mechanical properties of the ECM regulate focal adhesion and F-actin morphology as well as numerous physiological processes, including cell differentiation, division, proliferation and migration. Thus, the use of cell culture substrates has become an increasingly prevalent method to precisely control and modulate ECM mechanical properties.
To quantify traction forces at focal adhesions in an adherent cell, compliant substrates are used in conjunction with high-resolution imaging and computational techniques in a method termed traction force microscopy (TFM). This technique relies on measurements of the local magnitude and direction of substrate deformations induced by cellular contraction. In combination with high-resolution fluorescence microscopy of fluorescently tagged proteins, it is possible to correlate cytoskeletal organization and remodeling with traction forces.
Here we present a detailed experimental protocol for the preparation of two-dimensional, compliant matrices for the purpose of creating a cell culture substrate with a well-characterized, tunable mechanical stiffness, which is suitable for measuring cellular contraction. These protocols include the fabrication of polyacrylamide hydrogels, coating of ECM proteins on such gels, plating cells on gels, and high-resolution confocal microscopy using a perfusion chamber. Additionally, we provide a representative sample of data demonstrating location and magnitude of cellular forces using cited TFM protocols.

In this video, we demonstrate the experimental techniques used to fabricate compliant, extracellular matrix (ECM) coated substrates suitable for cell culture, and which are amenable to traction force microscopy and observing effects of ECM stiffness on cell behavior.

Preparación de las matrices de demanda para la cuantificación de la contracción celular

The regulation of cellular adhesion to the extracellular matrix (ECM) is essential for cell migration and ECM remodeling.  Focal adhesions are ...

Investigación

Bioingeniería

Fabrication of Micro-tissues using Modules of Collagen Gel Containing Cells

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized tissues.  The modules can be made of collagen as well as other gelable or crosslinkable materials.  They are approximately 2 mm in length and 0.7 mm in diameter upon fabrication but shrink in size with embedded cells or when the modules are coated with endothelial cells.  The modules individually are small enough that the embedded cells are within the diffusion limit of oxygen and other nutrients but modules can be packed together to form larger tissues that are perfusable.  These tissues are modular in construction because different cell types can be embedded in or coated on the modules before they are packed together to form complex tissues.  There are three main steps to making the modules: (1) neutralizing the collagen and embedding cells in it, (2) gelling the collagen in the tube and cutting the modules and (3) coating the modules with endothelial cells.

Creation of micro-tissues using cylindrical collagen gels, called modules, that contain embedded cells and which surface is coated with endothelial cells.

Fabricación de micro-tejidos, utilizando los módulos de gel de colágeno las células que contienen

This protocol describes the fabrication of a type of micro-tissues called modules.  The module approach generates uniform, scalable and vascularized ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

La encapsulación de los cardiomiocitos en un hidrogel de fibrina para ingeniería de tejidos cardíacos

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparación de fibrina Andamios 3D para Aplicaciones madre para cultivo celular

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

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

Collagen content and organization in developing collagenous tissues can be influenced by local tissue strains and tissue constraint. Tissue engineers aim to use these principles to create tissues with predefined collagen architectures. A full understanding of the exact underlying processes of collagen remodeling to control the final tissue architecture, however, is lacking. In particular, little is known about the (re)orientation of collagen fibers in response to changes in tissue mechanical loading conditions. We developed an in&#160;vitro model system, consisting of biaxially-constrained myofibroblast-seeded fibrin constructs, to further elucidate collagen (re)orientation in response to i) reverting biaxial to uniaxial static loading conditions and ii) cyclic uniaxial loading of the biaxially-constrained constructs before and after a change in loading direction, with use of the Flexcell FX4000T loading device. Time-lapse confocal imaging is used to visualize collagen (re)orientation in a nondestructive manner.
Cell and collagen organization in the constructs can be visualized in real-time, and an internal reference system allows us to relocate cells and collagen structures for time-lapse analysis. Various aspects of the model system can be adjusted, like cell source or use of healthy and diseased cells. Additives can be used to further elucidate mechanisms underlying collagen remodeling, by for example adding MMPs or blocking integrins. Shape and size of the construct can be easily adapted to specific needs, resulting in a highly tunable model system to study cell and collagen (re)organization.

This model system starts from a myofibroblast-populated fibrin gel that can be used to study endogenous collagen (re)organization real-time in a nondestructive manner. The model system is very tunable, as it can be used with different cell sources, medium additives, and can be adapted easily to specific needs.

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 ...

Characterization of Leukocyte-platelet Rich Fibrin, A Novel Biomaterial

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral surgery. Unlike platelet rich plasma (PRP) that is a gel or a suspension, Leukocyte-Platelet Rich Fibrin (L-PRF) is a solid 3D fibrin membrane generated chair-side from whole blood containing no anti-coagulant. The membrane has a dense three dimensional fibrin matrix with enriched platelets and abundant growth factors. L-PRF is a popular adjunct in surgeries because of its superior handling characteristics as well as its suturability to the wound bed. The goal of the study is to demonstrate generation as well as provide detailed characterization of relevant properties of L-PRF that underlie its clinical success.

Leucocyte-Platelet Rich Fibrin (L-PRF) represents an FDA cleared preparation of autologous platelet concentrates that possesses unique fibrin architecture, enriched platelets and abundant growth factors. Here, we present a protocol for chair-side generation of L-PRF as well as evaluate its mechanical properties including uniaxial testing and suture retention strength testing.

Caracterización de los leucocitos-plaquetas Rich fibrina, una novela de Biomateriales

Autologous platelet concentrates represent promising innovative tools in the field of regenerative medicine and have been extensively used in oral ...

Forming Giant-sized Polymersomes Using Gel-assisted Rehydration

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier properties, chemical versatility and tunable physical characteristics. Typical methods used to prepare giant-sized (&gt; 4 &#181;m) vesicles, however, are both time and labor intensive, yielding low numbers of intact polymersomes. Here, we present for the first time the use of gel-assisted rehydration for the rapid and high-yielding formation of giant (&gt;4 &#181;m) polymer vesicles (polymersomes). Using this method, polymersomes can be formed from a wide array of rehydration solutions including several different physiologically-compatible buffers and full cell culture media, making them readily useful for biomimicry studies. This technique is also capable of reliably producing polymersomes from different polymer compositions with far better yields and much less difficulty than traditional methods. Polymersome size is readily tunable by altering temperature during rehydration or adding membrane fluidizers to the polymer membrane, generating giant-sized polymersomes (&gt;100 &#181;m).

We present a protocol to rapidly form giant polymer vesicles (pGVs). Briefly, polymer solutions are dehydrated on dried agarose films adhered to coverslips. Rehydration of the polymer films results in rapid formation of pGVs. This method greatly advances the preparation of synthetic giant vesicles for direct applications in biomimetic studies.

Formando polímerosomas de tamaño gigante Uso de rehidratación asistida-Gel

Polymer vesicles, or polymersomes, are being widely explored as synthetic analogs of lipid vesicles based on their stability, robustness, barrier ...

A Novel Technique for Generating and Observing Chemiluminescence in a Biological Setting

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are quickly moving into the operating room. Here, we present a new approach that utilizes a technique not yet explored for intraoperative imaging: chemiluminescent imaging. This method employs a ruthenium-based chemiluminescent reporter along with a custom-built nebulizing system to produce ex vivo or in vivo images with high signal-to-noise ratios. The ruthenium-based reporter produces light following exposure to an aqueous oxidizing solution and re-reduction within the surrounding tissue. This method has allowed us to detect reporter concentrations as low as 6.9 pmol/cm2. In this work, we present a visual guide to our proof-of-concept in vivo studies involving subdermal and intravenous injections in mice. The results suggest that this technology is a promising candidate for further preclinical research and might ultimately become a useful tool in the operating room.

This protocol describes a new intraoperative imaging technique that uses a ruthenium complex as a source of chemiluminescent light emission, thereby producing high signal-to-noise ratios during in vivo imaging. Intraoperative imaging is an expanding field that could revolutionize the way that surgical procedures are performed.

Una nueva técnica para generar y Observación de quimioluminiscencia en un Contexto biológico

Intraoperative imaging techniques have the potential to make surgical interventions safer and more effective; for these reasons, such techniques are ...

A Microfluidic Flow Chamber Model for Platelet Transfusion and Hemostasis Measures Platelet Deposition and Fibrin Formation in Real-time

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is restricted to platelet deposition only. The intricate relationship between Ca2+-dependent coagulation and platelet function requires careful and controlled recalcification of blood prior to analysis. Our setup uses a Y-shaped mixing channel, which supplies concentrated Ca2+/Mg2+ buffer to flowing blood just prior to perfusion, enabling rapid recalcification without sample stasis. A ten-fold difference in flow velocity between both reservoirs minimizes dilution. The recalcified blood is then perfused in a collagen-coated analysis chamber, and differential labeling permits real-time imaging of both platelet and fibrin deposition using fluorescence video microscopy. The system uses only commercially available tools, increasing the chances of standardization. Reconstitution of thrombocytopenic blood with platelets from banked concentrates furthermore models platelet transfusion, proving its use in this research domain. Exemplary data demonstrated that coagulation onset and fibrin deposition were linearly dependent on the platelet concentration, confirming the relationship between primary and secondary hemostasis in our model. In a timeframe of 16 perfusion min, contact activation did not take place, despite recalcification to normal Ca2+ and Mg2+ levels. When coagulation factor XIIa was inhibited by corn trypsin inhibitor, this time frame was even longer, indicating a considerable dynamic range in which the changes in the procoagulant nature of the platelets can be assessed. Co-immobilization of tissue factor with collagen significantly reduced the time to onset of coagulation, but not its rate. The option to study the tissue factor and/or the contact pathway increases the versatility and utility of the assay.

This paper describes an experimental model of hemostasis that simultaneously measures platelet function and coagulation. Platelet and fibrin fluorescence is measured in real-time, and platelet adhesion rate, coagulation rate, and onset of coagulation are determined. The model is used to determine platelet procoagulant properties under flow in concentrates for transfusion.

Una Cámara de microfluidos Modelo de Flujo de Transfusión de Plaquetas y hemostasia Medidas de deposición de plaquetas y la formación de fibrina en tiempo real

Microfluidic models of hemostasis assess platelet function under conditions of hydrodynamic shear, but in the presence of anticoagulants, this analysis is ...

Gel-seq: A Method for Simultaneous Sequencing Library Preparation of DNA and RNA Using Hydrogel Matrices

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the standard protocols for generating genomic or transcriptomic libraries are incompatible and researchers must choose whether to sequence DNA or RNA for a particular sample. Gel-seq solves this problem by enabling researchers to simultaneously prepare libraries for both DNA and RNA starting with 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries. We designed Gel-seq so that it could be easily implemented by other researchers; many genetics labs already have the necessary equipment to reproduce the Gel-seq device fabrication. Our protocol employs commonly-used kits for both whole-transcript amplification (WTA) and library preparation, which are also likely to be familiar to researchers already versed in generating genomic and transcriptomic libraries. Our approach allows researchers to bring to bear the power of both DNA and RNA sequencing on a single sample without splitting and with negligible added cost.

Gel-seq enables researchers to simultaneously prepare libraries for both DNA- and RNA-seq at negligible added cost starting from 100 - 1000 cells using a simple hydrogel device. This paper presents a detailed approach for the fabrication of the device as well as the biological protocol to generate paired libraries.

Gel-seq: Un método para la preparación de biblioteca simultánea de secuenciación de DNA y RNA utilizando Matrices de hidrogel

The ability to amplify and sequence either DNA or RNA from small starting samples has only been achieved in the last five years. Unfortunately, the ...