Este proyecto fue financiado por la Swiss National Science Foundation (SNF # 310030-127586/1).

1. La cosecha del disco intervertebral <ol><li> La cola toda la longitud de la especie bovina se obtiene de un matadero local, si es posible sin la piel ya que la presencia de la piel aumenta la probabilidad de contaminación (Fig. 2). </li><li> Prepare la tabla de cortar grande y preparar la estación de trabajo e instrumentos estériles en la parte superior de una tabla de cortar (Fig. 2). </li><li> Preparar bajo la capucha de gasa estéril laminar flujo estéril humedecida con cloruro sódico al 0,9% que contiene citrato de sodio de 55 mm y poner en cada pocillo de la placa de 6 pocillos. </li><li> Prepare un recipiente y se diluye 1:100 solución de Betadine con agua del grifo. </li><li> Sumerja la cola de la especie bovina todo en un recipiente que contiene 1% de solución de Betadine durante 5 min. </li><li> En pocas palabras la cola seca con una gasa estéril y colocarlo en la estación de trabajo estéril. </li><li> Retire con cuidado los músculos alrededor de la cola con hoja de bisturí # 22. </li><li> Tajan las partes de la cola que no son necesarios, por lo general los dos extremos de la cola, que contienen relativamente grandes y muy pequeñosDIV (Fig. 3). </li><li> Recortar aún más los músculos y tendones alrededor de la DIV con hoja de bisturí # 10. Tenga cuidado de no cortar el anillo externo de los discos. </li><li> Marque la parte anterior del IVD con un marcador quirúrgico de la piel (este paso es opcional, que ayuda a determinar el centro de la rotación axial de nuestro biorreactor). </li><li> Localice el IVD y el punto de conexión vértebra moviendo la cola suavemente. Siente la frontera entre IVD y el hueso con el lado opaco de una hoja de bisturí. El sitio de escisión debe ser 1-2 mm de distancia del IVD a la vértebra. Personalizada lugar hizo poseedor de la hoja industrial en el sitio de clivaje (Fig. 4). </li><li> Cleave IVD y una vértebra a golpe de martillo en la parte superior del soporte de la cuchilla a medida industrial (Fig. 3). </li><li> Repita en el otro lado de la conexión del DIV-vértebra. </li><li> Envuelva aislados IVD con una gasa estéril humedecida con cloruro sódico al 0,9% que contiene citrato de sodio de 55 mm. </li><li> Continuar con el aislamiento de DIV en el número deseado de la muestra (por lo general alrededor de 6 puedeser cosechados con un diámetro entre 10-20 mm). </li><li> Conecte el sistema de lavado de chorro (Zimmer, inc.) Con solución de Ringer lactato estéril, 5L bolsas son de un tamaño útil (como alternativa PBS estéril o solución salina 0,9%). </li><li> Sostenga el IVD con pinzas y el lavado de chorro-los dos lados de la superficie de la placa final con Zimmer Pulsavac chorro de lavado del sistema (Fig. 1C). La pistola de chorro se debe tirar en la superficie de la placa terminal en un ángulo entre 30 a 60 ° durante unos 30 a cada lado. (Figuras 1 y 7) </li><li> Ponga el DIV de nuevo a la placa de 6 pocillos y envolver con una gasa humedecida, mientras que otra muestra de chorro de lavado de disco. </li><li> DIV están listos para el cultivo de órganos y aguas abajo de aplicaciones (por ejemplo, viabilidad celular, la carga mecánica, la cultura de órganos) (Fig. 1D). </li></ol> 2. Resultados representante Mediciones de los resultados para juzgar a una preparación exitosa del disco intervertebral puede ser un experimento de difusión, donde un tinte fluorescente de bajo peso molecular (por ejemplo, Procion rojo)se prepara como una solución al 1% en PBS 2. El IVD es luego mantenido con al menos 100 ml de solución de tinte y tinte entonces se permite difundir pasivamente en el IVD durante 24 horas bajo la hinchazón libre. El IVD es entonces flash-congeladas en nitrógeno líquido y trajo de vuelta a la temperatura ambiente y se deshidrata por una serie de transferencias de acetona (primera pre-enfriado de -80 ° C, entonces -20 ° C, luego 4 ° C y, finalmente, a la temperatura ambiente ). El disco puede ser embebido en poli-metil-acrilato de metilo (PMMA) y cortar con una cuchilla afilada para producir 100 micras de espesor secciones. Estas secciones se pueden ver por cualquier microscopio de campo claro estándar, pero de preferencia por el uso de microscopía de fluorescencia desde Procion rojo emite una fluorescencia de color rojo (Fig. 5-6). El trabeculi de la superficie ósea parece muy limpio después del paso de lavaging jet (Fig. 7). Los parámetros de una cultura de órganos exitosos son en primer lugar la ausencia de contaminación, mantener la viabilidad celular (Figs. 8-9), la altura del disco ygeneral &quot;del disco integridad&quot;, medida con cortes histológicos y safranina O / verde o tinción Hematoxilina de Meyer 3. Hemos desarrollado protocolos y macros publicado en otra parte 4,5 para teñir el tejido del disco en vivo y para explorar la viabilidad celular en 3D usando un microscopio láser confocal de barrido y el kit de coloración en vivo / muerto (Molecular Probes, Invitrogen, Basilea, Suiza). Hemos desarrollado más de una macro y la rutina para realizar conteo de células automatizado para estimar la viabilidad celular en el tejido 3D o 3D los transportistas que utilizan la plataforma NIH ImageJ 4-6. Protocolos alternativos han sido propuestos para determinar la viabilidad celular de las células del disco por la digestión inicial mediante digestión secuencial de la matriz extracelular con pronasa y colagenasa tipo 2 y luego a la mancha de la suspensión celular 7. Una versión anterior del protocolo ha supuesto un paso de incubación en PBS que contenía diez veces más concentrado concentración de penicilina / estreptomicina durante 10 minutos después de la recolección de los discos antes de órganos culture. Con el paso de lavado de chorro de este paso se convierte en obsoleto ya que este paso parece beneficioso para fines de cultivo. <img alt="Figura 1" src="/files/ftp_upload/3490/3490fig1.jpg" /> Figura 1. Descripción de los pasos metodológicos para preparar bovina discos intervertebrales coccígea con placas intactas y una capa fina (1-2mm) de los huesos. A) Representación esquemática del disco intervertebral (DIV) y sus vías de nutrición. B) Procedimiento para cortar el IVD con una hoja personalizada del titular y una cuchilla afilada industrial. C) Jet lavado paso para asegurar la viabilidad celular alta para permitir el intercambio de nutrientes mantener las placas terminales y 1-2 mm de hueso adjunto a ambos lados. D) Por último, el IVD pueden ser cultivados en la cámara de muestra de un biorreactor o ser utilizado para cualquier aplicación de aguas abajo. <img alt="Figura 2" src="/files/ftp_upload/3490/3490fig2.jpg" /> Figura 2 TOP:. Longitud total fresca de cola de la especie bovina (idealmente obtaINED a las 2-3 horas post-mortem para asegurar la viabilidad celular alta. Abajo: imagen de rayos X de una cola de la especie bovina de 6 meses de edad que ilustra la existencia de placas de crecimiento (GP), un centro secundario de osificación, entre las placas extremas ósea (EP) y los cuerpos de vértebras (VB). <img alt="Figura 3" src="/files/ftp_upload/3490/3490fig3.jpg" /> Figura 3. A Tools) para diseccionar la cola de la especie bovina en condiciones estériles. B) a medida el titular de la hoja de corte de los segmentos promocionales de la cola de la especie bovina en la acción, mientras que el corte del disco intervertebral. C) Vista lateral de la hoja personalizada titular con una hoja estándar industrial (Lutz, Alemania). <img alt="Figura 4" src="/files/ftp_upload/3490/3490fig4.jpg" /> Figura 4. Dibujo y foto de la cola de la especie bovina después de retirar el tejido circundante y ligamentos que ilustran los sitios de escisión para cortar los segmentos promocionales. Parcial vértebra debe ser &quot;agrietado&quot; wiª del soporte de la cuchilla a medida idealmente 1-2 mm de distancia de las placas terminales cartilaginosas para asegurar que la superficie ósea completa. <img alt="Figura 5" src="/files/ftp_upload/3490/3490fig5.jpg" /> Figura 5. Demostración de la limpieza efectiva de la etapa de jet-lavado antes (A) y después (B). Observe las superficies limpias ósea de los segmentos de disco intervertebral después de que el procedimiento de fumigación. <img alt="Figura 6" src="/files/ftp_upload/3490/3490fig6.jpg" /> Figura 6. Free-inflamación experimento difusión de recién preparada extirpado los discos intervertebrales cola bovina (IVD) a la izquierda por 24 horas en una solución de 1% Procion rojo, lo que demuestra efecto de bloqueo de la placa de crecimiento. Una radiografía digital sagital de un DIV que todavía contiene la placa de crecimiento y una segunda capa de la imagen de microscopía de fluorescencia de hueso corte sagital a través de la coccígea explante bovina disco intervertebral con placa terminal cartilaginosa y ósea después de 24h libre difusión. GP: placa de crecimiento, 2ndry CO: centro secundario de osificación. <img alt="Figura 7" src="/files/ftp_upload/3490/3490fig7.jpg" /> Figura 7. Free-inflamación experimento de recién preparada extirpado los discos intervertebrales cola bovina (IVD) a la izquierda por 24 horas en una solución de 1% Procion rojo, lo que demuestra el efecto del tratamiento de limpieza de chorro de lavado. Sagital secciones gruesas (~ 100 mm) de extirpar DIV bovina con placas de ~ 1,5 mmthick final ósea que se sometieron a un tratamiento de chorro de lavado (izquierda) el control sin tratamiento (derecha) jet-lavaged tratamiento. De entrada muestra el patrón de spray, que fue utilizado por la herida Zimmer desbridamiento sistema experimentos 24h libre difusión de bovinos explantes de disco intervertebral en rojo Procion 1%. <img alt="Figura 8" src="/files/ftp_upload/3490/3490fig8.jpg" /> Figura 8. Imágenes Live / Dead de proyecciones de imágenes confocal pilas (250 micras) tomadas de bovinsegmentos e disco intervertebral preparados con este método de chorro de lavado mantenido bajo libre inflamación de los días 0 y 14, respectivamente, para el núcleo pulposo (NP), el interior del anillo fibroso (IA) y el anillo fibroso externo (OA). Las células verde = células vivas manchado por calceína AM (acetil éster metílico), glóbulos rojos = núcleo de las células muertas teñidas con homodímero de etidio-1. <img alt="Figura 9" src="/files/ftp_upload/3490/3490fig9.jpg" /> Figura 9. Viabilidad de las células del disco intervertebral con láser confocal de barrido microscopio en los tejidos vivos y escaneo 3D pila. La viabilidad celular de núcleo pulposo (NP) y anillo fibroso (AF) en los 21 días de cultivo bajo condiciones de libre hinchazón. (Media ± SEM, n = 6) Las diferencias estadísticas se sometieron a pruebas no paramétricas de Kruskal-Wallis firmado prueba la suma de rangos entre los grupos. Se encontraron diferencias significativas entre los días 21 y todos los puntos de otro momento de la NP. Mientras que en las diferencias de AF se encontraron entre el día 7 deel día 0. (* P &lt;0,05, ** p &lt;0,01).

<ol>
	<li>Lee, C. R., Iatridis, J. C., Poveda, L., Alini, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+organ+culture+of+the+bovine+intervertebral+disc:+effects+of+vertebral+endplate+and+potential+for+mechanobiology+studies.">In vitro organ culture of the bovine intervertebral disc: effects of vertebral endplate and potential for mechanobiology studies.</a> Spine (Phila Pa 1976). 31, 515-522 (1976).</li><li>Chan, S. C. W., Gantenbein-Ritter, B., Leung, V. Y., Chan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cryopreserved+intervertebral+disc+with+injected+bone+marrow-derived+stromal+cells:+a+feasibility+study+using+organ+culture.">Cryopreserved intervertebral disc with injected bone marrow-derived stromal cells: a feasibility study using organ culture.</a> Spine. J. 10 (6), 486-496 (2010).</li><li>Chan, S. C., Ferguson, S. J., Wuertz, K., Gantenbein-Ritter, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+Response+of+the+Intervertebral+Disc+to+Repetitive+Short+Term+Cyclic+Torsion.">Biological Response of the Intervertebral Disc to Repetitive Short Term Cyclic Torsion.</a> Spine (Phila Pa 1976). , (2011).</li><li>Gantenbein-Ritter, B., Sprecher, C. M., Chan, S., Illien-J&#252;nger, S., Grad, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Confocal+imaging+protocols+for+live/dead+staining+in+three-dimensional+carriers.">Confocal imaging protocols for live/dead staining in three-dimensional carriers.</a> Methods Mol. Biol. 740, 127-140 (2011).</li><li>Gantenbein-Ritter, B., Potier, E., Zeiter, S., van der Werf, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accuracy+of+three+techniques+to+determine+cell+viability+in+3D+tissues+or+scaffolds.">Accuracy of three techniques to determine cell viability in 3D tissues or scaffolds.</a> Tissue Engineering Part C Methods. 14, 353-358 (2008).</li><li>Rasband, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> ImageJ. , (1997).</li><li>Haschtmann, D., Stoyanov, J. V., Ettinger, L., Nolte, L. P., Ferguson, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishment+of+a+novel+intervertebral+disc/endplate+culture+model:+analysis+of+an+ex+vivo+in+vitro+whole-organ+rabbit+culture+system.">Establishment of a novel intervertebral disc/endplate culture model: analysis of an ex vivo in vitro whole-organ rabbit culture system.</a> Spine. 31, 2918-2925 (2006).</li><li>Gantenbein, B., Gr&#252;nhagen, T., Lee, C. R., van Donkelaar, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vitro+organ+culturing+system+for+intervertebral+disc+explants+with+vertebral+endplates:+a+feasibility+study+with+ovine+caudal+discs.">An in vitro organ culturing system for intervertebral disc explants with vertebral endplates: a feasibility study with ovine caudal discs.</a> Spine. 31, 2665-2673 (2006).</li><li>Korecki, C. L., Kuo, C. K., Tuan, R. S., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intervertebral+disc+cell+response+to+dynamic+compression+is+age+and+frequency+dependent.">Intervertebral disc cell response to dynamic compression is age and frequency dependent.</a> J. Orthop. Res. 27, 800-806 (2009).</li><li>Korecki, C. L., MacLean, J. J., Iatridis, J. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+compression+effects+on+intervertebral+disc+mechanics+and+biology.">Dynamic compression effects on intervertebral disc mechanics and biology.</a> Spine (Phila Pa 1976). 33, 1403-1409 (1976).</li><li>Jim, B., Steffen, T., Moir, J., Roughley, P., Haglund, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+intact+intervertebral+disc+organ+culture+system+in+which+degeneration+can+be+induced+as+a+prelude+to+studying+repair+potential.">Development of an intact intervertebral disc organ culture system in which degeneration can be induced as a prelude to studying repair potential.</a> European Spine Journal. , 1-11 (2011).</li></ol>

No tenemos nada que revelar.

El primer paso para el cultivo de órganos con éxito es asegurarse de que el explante no debe estar contaminada. La cola debe ser de piel antes de comenzar con el procedimiento. Cualquier pelo llevados a un laboratorio estéril podría ser problemático en términos de contaminación. La cola de la especie bovina idealmente deben estar tan frescos como sea posible (esto afecta a la viabilidad inicial de la célula). Además, el paso de lavado betadine se recomienda para reducir el riesgo de contaminación. En lugar de ...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td colspan="4"> De tejido fresco de bovino disco intervertebral de las colas de las especies bovina, de la casa de masacre local (idealmente dentro de las horas post-mortem y sin piel). </td></tr><tr><td> Pulsavac Plus System CA </td><td> Zimmer inc., Suiza </td><td> 00-5150-486-01 </td><td> El mejor rendimiento con el jefe de la cadera en aerosol y con fuente de alimentación de corriente alterna (la que tiene el paquete de 8 pilas AA también funciona, pero es menos conveniente) </td></tr><tr><td> Alta capacidad del ventilador spray w / Protector contra salpicaduras, longitud 12,7 cm </td><td> Zimmer inc., Suiza </td><td> 00-5150-175-00 </td><td> Hay varias cabezas de aerosol disponibles, hemos probado con éxito este </td></tr><tr><td> Hojas de bisturí # 22 y # 10 </td><td> Swann-Morten, Inglaterra </td><td> # 10: 0201 # 22: 0208 </td><td></td></tr><tr><td> Titular de la hoja de bisturí # 3 y # 4 </td><td> Hausmann, Alemania </td><td> # 3: 06.103.00 # 4: 06.104.00 </td><td></td></tr><tr><td> Lutz industrial blade </td><td> Lutz, Alemania </td><td> 1022.0884 </td><td></td></tr><tr><td> Tampón fosfato salino (PBS) </td><td> Invitrogen, Suiza </td><td> 10010-023 </td><td></td></tr><tr><td> Dulbecco Eagle modificado (DMEM) </td><td> Gibco, Suiza </td><td> 11960-044 </td><td></td></tr><tr><td> Solución de Ringer lactato (sin glucosa) </td><td> Bichsel, Suiza </td><td> 133 0002 </td><td></td></tr><tr><td> 6-y multi-pocillos </td><td> TPP, Suiza </td><td> 92006 </td><td></td></tr><tr><td> Betadine solución </td><td> Mundipharma, Suiza </td&gt; <td> 10055025 </td><td></td></tr><tr><td> Marcador quirúrgica de la piel </td><td> Porex Surgical, Suiza </td><td> 9560 </td><td></td></tr><tr><td> Tabla de cortar grande </td><td></td><td></td><td> Cualquier marca es posible </td></tr></tbody></table>

preparation of intact bovine tail intervertebral discs for organ culture

El disco intervertebral (DIV) es la articulación de la columna vértebra a vértebra conexión. Su función es transmitir la carga de la columna y dar flexibilidad a la columna vertebral. Se compone de tres compartimentos: el núcleo pulposo interior (NP), que abarca el anillo fibroso (AF), y dos placas de extremo cartilaginoso conecta el PN y el FA en el cuerpo vertebral en ambos lados. El dolor discogénico, posiblemente causado por una enfermedad degenerativa del disco intervertebral (DDD) y hernias de disco ha sido identificado como un problema importante en nuestra sociedad moderna. Para estudiar los posibles mecanismos de la degeneración IVD, en los sistemas de cultivo in vitro con células de órganos disco en vivo son muy atractivas. El cultivo in vitro de la especie bovina intacta DIV coccígea ha avanzado a un sistema de modelo correspondiente, que permite el estudio de la mecano-biológicos aspectos en un ambiente bien controlado fisiológicos y mecánicos. DIV bovina cola se puede obtener con relativa facilidad en un mayor númerod son muy similares a los humanos DIV lumbar con respecto a la densidad celular, la población de células y las dimensiones. Sin embargo, las técnicas anteriores bovina caudal cosecha IVD retener platillos cartilaginosos y óseos platillos no después de 1-2 días de cultivo ya que las vías fueron bloqueadas, obviamente, la nutrición por la sangre coagulada. DIV son los principales órganos avascular, por lo tanto, los nutrientes a las células en el PN sólo dependen de la difusión a través de las papilas capilares del cuerpo vertebral adyacente. Presencia de restos de huesos y la sangre coagulada en la superficie de la placa terminal puede impedir la difusión de nutrientes en el centro del disco y la viabilidad celular compromiso. Nuestro grupo ha establecido un protocolo relativamente rápida de &quot;crack&quot;, el DIV de la cola con un bajo riesgo de contaminación. Somos capaces de permeabilizar las superficies óseas recién cortada placa terminal mediante un sistema de chorro de lavado quirúrgico, que elimina los coágulos de sangre y restos de corte y de manera muy eficiente se vuelve a abrir la vía de difusión de nutriciónen el centro de la DIV. La presencia de placas de crecimiento en ambos lados del hueso vertebral tiene que ser evitada y para ser removido antes de la cultura. En este vídeo, se exponen los pasos cruciales en la preparación y demostrar la clave para una cultura de órganos con éxito el mantenimiento de la viabilidad celular alta durante 14 días en cultivo sin inflamación. El tiempo de cultivo podría extenderse al entorno mecánico adecuado se puede mantener mediante el uso de biorreactores de carga mecánica. La técnica ha demostrado aquí se puede extender a otras especies animales, como porcinos, ovinos y aislamiento leporino IVD caudal y lumbar.

Watch this Scientific Journal Video about Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture at JoVE.com

Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

Este protocolo ilustra una técnica de recolección de coccígea discos intervertebrales de la especie bovina para el cultivo de órganos para<em&gt; In vitro</em&gt; La cultura de órganos.

Preparación de la cola intacta discos intervertebrales bovina para la Cultura de Órganos

The intervertebral disc (IVD) is the joint of the spine connecting  vertebra to vertebra. It functions to transmit loading of the spine and give  ...

preparation-intact-bovine-tail-intervertebral-discs-for-organ

Research

JoVE Journal

Bioengineering

14.4K vistas.  University of Bern. Este protocolo ilustra una técnica de recolección de coccígea discos intervertebrales de la especie bovina para el cultivo de órganos para In vitro La cultura de órganos.

Video: Preparation of Intact Bovine Tail Intervertebral Discs for Organ Culture

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

Alginate Hydrogels for Three-Dimensional Organ Culture of Ovaries and Oviducts

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ovarian cancers is still in question and might be either the ovarian surface epithelium (OSE) or the distal epithelium of the fallopian tube fimbriae2,3. Culturing the normal cells as a primary culture in vitro will enable scientists to model specific changes that might lead to ovarian cancer in the distinct epithelium, thereby definitively determining the cell type of origin. This will allow development of more accurate biomarkers, animal models with tissue-specific gene changes, and better prevention strategies targeted to this disease.
Maintaining normal cells in alginate hydrogels promotes short term in vitro culture of cells in their three-dimensional context and permits introduction of plasmid DNA, siRNA, and small molecules. By culturing organs in pieces that are derived from strategic cuts using a scalpel, several cultures from a single organ can be generated, increasing the number of experiments from a single animal. These cuts model aspects of ovulation leading to proliferation of the OSE, which is associated with ovarian cancer formation. Cell types such as the OSE that do not grow well on plastic surfaces can be cultured using this method and facilitate investigation into normal cellular processes or the earliest events in cancer formation4.
Alginate hydrogels can be used to support the growth of many types of tissues5. Alginate is a linear polysaccharide composed of repeating units of &beta;-D-mannuronic acid and &alpha;-L-guluronic acid that can be crosslinked with calcium ions, resulting in a gentle gelling action that does not damage tissues6,7. Like other three-dimensional cell culture matrices such as Matrigel, alginate provides mechanical support for tissues; however, proteins are not reactive with the alginate matrix, and therefore alginate functions as a synthetic extracellular matrix that does not initiate cell signaling5. The alginate hydrogel floats in standard cell culture medium and supports the architecture of the tissue growth in vitro.
A method is presented for the preparation, separation, and embedding of ovarian and oviductal organ pieces into alginate hydrogels, which can be maintained in culture for up to two weeks. The enzymatic release of cells for analysis of proteins and RNA samples from the organ culture is also described. Finally, the growth of primary cell types is possible without genetic immortalization from mice and permits investigators to use knockout and transgenic mice.

Culture of normal cells in their three-dimensional context represents an alternative method to study early events required for cellular transformation and tumorigenesis. This method is used to grow normal ovarian and oviductal cells to study early events in ovarian cancer formation.

Los hidrogeles de alginato para la cultura del órgano en tres dimensiones de los ovarios y oviductos

Ovarian cancer is the fifth leading cause of cancer deaths in women and has a 63% mortality rate in the United States1. The cell type of origin for ...

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 ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Preparación de la muestra Estrategias para Espectrometría de Masas de imágenes de modelos de cultivo celular en 3D

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

The Multi-organ Chip - A Microfluidic Platform for Long-term Multi-tissue Coculture

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need for new solutions for substance testing. Many drugs that have been removed from the market due to drug-induced liver injury released their toxic potential only after several doses of chronic testing in humans. However, a controlled microenvironment is pivotal for long-term multiple dosing experiments,&#160;as even minor alterations in extracellular conditions may greatly influence the cell physiology. We focused within our research program on the generation of a microengineered bioreactor, which can be dynamically perfused by an on-chip pump and combines at least two culture spaces for multi-organ applications. This circulatory system mimics the in vivo conditions of primary cell cultures better and assures a steadier,&#160;more quantifiable extracellular relay of signals to the cells. 
For demonstration purposes, human liver equivalents, generated by aggregating differentiated HepaRG cells with human hepatic stellate cells in hanging drop plates, were cocultured with human skin punch biopsies for up to 28 days inside the microbioreactor. The use of cell culture inserts enables the skin to be cultured at an air-liquid interface, allowing topical substance exposure. The microbioreactor system is capable of supporting these cocultures at near physiologic fluid flow and volume-to-liquid ratios, ensuring stable and organotypic culture conditions. The possibility of long-term cultures enables the repeated exposure to substances. Furthermore, a vascularization of the microfluidic channel circuit using human dermal microvascular endothelial cells yields a physiologically more relevant vascular model.

Here, we present a protocol to coculture primary cells, tissue models and punch biopsies in a microfluidic multi-organ chip for up to 28 days. Human dermal microvascular endothelial cells, liver aggregates and skin biopsies were successfully combined in a common media circulation.

El chip de varios órganos - Una plataforma de microfluidos para el largo plazo co-cultivo de múltiples tejidos

The ever growing amount of new substances released onto the market and the limited predictability of current in vitro test systems has led to a high need ...

Studying the Stoichiometry of Epidermal Growth Factor Receptor in Intact Cells using Correlative Microscopy

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence microscopy and environmental scanning electron microscopy (ESEM) of whole cells in hydrated state. Fluorescent quantum dots (QDs) were coupled to EGFR via a two-step labeling protocol, providing an efficient and specific protein labeling, while avoiding label-induced clustering of the receptor. Fluorescence microscopy provided overview images of the cellular locations of the EGFR. The scanning transmission electron microscopy (STEM) detector was used to detect the QD labels with nanoscale resolution. The resulting correlative images provide data of the cellular EGFR distribution, and the stoichiometry at the single molecular level in the natural context of the hydrated intact cell. ESEM-STEM images revealed the receptor to be present as monomer, as homodimer, and in small clusters. Labeling with two different QDs, i.e.,&#160;one emitting at 655 nm and at 800 revealed similar characteristic results.

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative light- and electron microscopy of whole cells in hydrated state. The label contained fluorescent quantum dots. The protocol can be used to study the stoichiometry of EGFR at the single molecule level.

El estudio de la estequiometría de factor de crecimiento epidérmico en células intactas utilizando microscopía correlativa

This protocol describes the labeling of epidermal growth factor receptor (EGFR) on COS7 fibroblast cells, and subsequent correlative fluorescence ...

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Nanoscaled sea-island surfaces composed of thermoresponsive block copolymers were fabricated by the Langmuir-Schaefer method for controlling spontaneous cell adhesion and detachment. Both the preparation of the surface and the adhesion and detachment of cells on the surface were visualized.

Preparación de Superficies de respuesta térmica nanoestructurados para la Ingeniería de Tejidos

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the ...

Preparation of Extracellular Matrix Protein Fibers for Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of visible light with thermally induced acoustic waves or phonons propagating at a speed of a few km/sec. Information on the elasticity and structure of the material is obtained in a nondestructive contactless manner, hence opening the way to in vivo applications and potential diagnosis of pathology. This work describes the application of Brillouin spectroscopy to the study of biomechanics in elastin and trypsin-digested type I collagen fibers of the extracellular matrix. Fibrous proteins of the extracellular matrix are the building blocks of biological tissues and investigating their mechanical and physical behavior is key to establishing structure-function relationships in normal tissues and the changes which occur in disease. The procedures of sample preparation followed by measurement of Brillouin spectra using a reflective substrate are presented together with details of the optical system and methods of spectral data analysis.

We present a protocol for the application of Brillouin light scattering spectroscopy to elastin and trypsin-purified type I collagen fibers of the extracellular matrix to extract their full elastic properties.

Preparación de la matriz extracelular fibras de proteína de Brillouin Spectroscopy

Brillouin spectroscopy is an emerging technique in the biomedical field. It probes the mechanical properties of a sample through the interaction of ...

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and dampen loads in the spine. The IVD consists of a proteoglycan-rich nucleus pulposus (NP) and a collagen-rich annulus fibrosis (AF) sandwiched by cartilaginous end-plates. Together with the adjacent vertebrae, the vertebrae-IVD structure forms a functional spine unit (FSU). These microstructures contain unique cell types as well as unique extracellular matrices. Whole organ culture of the FSU preserves the native extracellular matrix, cell differentiation phenotypes, and cellular-matrix interactions. Thus, organ culture techniques are particularly useful for investigating the complex biological mechanisms of the IVD. Here, we describe a high-throughput approach for culturing whole lumbar mouse FSUs that provides an ideal platform for studying disease mechanisms and therapies for the IVD. Furthermore, we describe several applications that utilize this organ culture method to conduct further studies including contrast-enhanced microCT imaging and three-dimensional high-resolution finite element modeling of the IVD.

An In Vitro Organ Culture Model of the Murine Intervertebral Disc

Whole organ culture of the intervertebral disc (IVD) preserves the native extracellular matrix, cell phenotypes, and cellular-matrix interactions. Here we describe an IVD culture system using mouse lumbar and caudal IVDs in their functional spinal units and several applications utilizing this system.

Un<em&gt; In Vitro</em&gt; Órgano Cultura Modelo Murino del disco intervertebral

Intervertebral disc (IVD) degeneration is a significant contributor to low back pain. The IVD is a fibrocartilaginous joint that serves to transmit and ...

Fabrication and Validation of an Organ-on-chip System with Integrated Electrodes to Directly Quantify Transendothelial Electrical Resistance

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful research tools for studying human health and disease. To characterize the barrier function of cell layers cultured inside organ-on-chip devices, often transendothelial or transepithelial electrical resistance (TEER) is measured. To this end, electrodes are usually integrated into the chip by micromachining methods to provide more stable measurements than is achieved with manual insertion of electrodes into the inlets of the chip. However, these electrodes frequently hamper visual inspection of the studied cell layer or require expensive cleanroom processes for fabrication. To overcome these limitations, the device described here contains four easily integrated electrodes that are placed and fixed outside of the culture area, making visual inspection possible. Using these four electrodes the resistance of six measurement paths can be quantified, from which the TEER can be directly isolated, independent of the resistance of culture medium-filled microchannels. The blood-brain barrier was replicated in this device and its TEER was monitored to show the device applicability. This chip, the integrated electrodes and the TEER determination method are generally applicable in organs-on-chips, both to mimic other organs or to be incorporated into existing organ-on-chip systems.

This publication describes the fabrication of an organ-on-chip device with integrated electrodes for direct quantification of transendothelial electrical resistance (TEER). For validation, the blood-brain barrier was mimicked inside this microfluidic device and its barrier function was monitored. The presented methods for electrode integration and direct TEER quantification are generally applicable.

Fabricación y validación de un sistema de órganos-en-viruta con electrodos integrados directamente cuantificar Transendothelial resistencia eléctrica

Organs-on-chips, in vitro models involving the culture of (human) tissues inside microfluidic devices, are rapidly emerging and promise to provide useful ...