Nos gustaría agradecer a Robin Nye (Tufts Medical Center), Uchimura Tomoya y Dana Cairns (Tufts University) para proporcionar ayuda para el almacenamiento del líquido sinovial y la centrifugación. Este trabajo fue financiado por el NIH (1R01AR059106-01A1) de LZ

Un sistema 3D para el cultivo de condrocitos articulares humanos en el líquido sinovial En este trabajo, se encapsulan los condrocitos humanos articular en perlas de alginato usando una versión modificada de fabricación-sugirió la encapsulación de protocolo (Lonza, y 31). El uso de estas construcciones en 3D, hemos desarrollado un sistema para el cultivo de células en un medio de cultivo que contienen porcentajes variaron de líquido sinovial humano y han evaluado estas construcciones 3D para la expresión de genes del cartílago. 1. Prepare los condrocitos humanos articular (HAC) de tres dimensiones (3D) en la encapsulación <ol><li> Descongelar un vial (1 ml) de los condrocitos articulares humanos (HAC) (Lonza) (paso 2) en 37 ° C baño de agua durante 1 minuto. </li><li> Mezclar con 1 ml de medio de cultivo de condrocitos (Lonza) y se centrifuga a 3000 rpm durante 3-5 minutos para recoger las células. Eliminar el sobrenadante. </li><li> Resuspender el sedimento celular en los medios de crecimiento de los condrocitos (Lonza). </li><li> Células de la placa en un tejido de 10 cmplaca de cultivo (BD Biosciences), y la cultura en los medios de crecimiento de los condrocitos (Lonza) hasta que las células son confluentes. HAC debe ser utilizado en un número de paso no superior a 3 o 4 (P3 o P4). </li><li> Lave HAC en la placa de 155 mm con NaCl en lugar de la PBS estándar, seguido por trypsinzation para recoger las células para la encapsulación de alginato de cuentas. Una vez que las células se separan, la desaceleración de las células a 3000 rpm durante 5 minutos. Las células están listas para la encapsulación en 3D. </li></ol> 2. Encapsular HAC en cuentas en 3D Resuspender el HAC en la solución de alginato de 1,2% (Sigma) a una densidad de 8x10 5 células / ml. El número de células se determinó antes de la encapsulación, utilizando un contador de células estándar. Es muy importante mezclar bien para asegurar una distribución uniforme de las células en las cuentas. <ol><li> Pipeta del HAC / mezcla de alginato solución a una jeringa de 12 ml conectada a una aguja de calibre 22 (Tyco Healthcare, Inc.). </li><li> Mientras tanto, prepare un vaso de 50 mL con 102 mM CaCl 2 a 5 veces el volumen de la solución de HAC / aginate. Coloque una barra de agitación en el interior del vaso y los 102 mM de CaCl2 solución lentamente (aproximadamente 150 rpm). </li><li> Sostenga la punta de la jeringa cerca de 6 pulgadas por encima de la superficie de la solución de CaCl2 y agregue la mezcla de HAC / alginato en el gota a gota una solución de CaCl 2. En general, las perlas de alginato resultantes son de 2 mm de diámetro con una densidad de encapsulación de aproximadamente 10 4 células / talón. Nota: La altura de la jeringa en la solución de CaCl2 es importante. La distancia más corta se traducirá en forma de gota en vez de esférica en forma de bolas, haciendo que la integridad mecánica desigual y la encapsulación de células. </li><li> Deje que las cuentas revuelo en la solución de CaCl2 durante 20-25 min. Mientras que otros protocolos de encapsulación alginato indican un tiempo de incubación mucho más corto de 10 minutos, se encontró que un mayor tiempo de incubación con la solución de CaCl2 mejorado la integridad de la seanuncios. </li><li> Retire la solución de CaCl2, lavar los granos 2-3 veces en 2-3 volúmenes de cloruro de sodio, y luego una vez en un medio de diferenciación de los condrocitos (Lonza). Mientras que otros protocolos de encapsulación alginato de cuentas involucran el uso de un filtro para los procedimientos anteriores de lavado, se encontró que las perlas de alginato a menudo quedan atrapados en el filtro y seco, lo que reduce la eficiencia de la encapsulación. Nos pareció que era más eficiente para permitir que los granos se depositen en el fondo del vaso antes de desechar la solución. </li><li> Utilice una espátula estándar para la transferencia de las cuentas en placas de cultivo. Por lo general, la cultura 12 cuentas por 3 cm y durante 2 días en medio de cultivo de condrocitos para permitir que los condrocitos para ajustar el proceso de encapsulación en el medio en que se cultivaron en medio antes de pasar a la diferenciación de condrocitos (Lonza) con el líquido sinovial. </li></ol> 3. Condrocitos de cultivo en medio líquido sinovial cultura <ol><li> El líquido sinovial fresco se puede obtener de una oclínica utpatient (Se obtuvo líquido sinovial de Tufts Medical Center). El líquido sinovial se puede transferir en tubos Falcon de 15 ml para el laboratorio, e inmediatamente se centrifuga a 3000 rpm durante 15 minutos para eliminar los restos celulares. Alícuota de células libres de líquido sinovial en tubos de 1,5 mL microcentrífuga, para evitar repetidos de congelación-descongelación. Sobrenadante se almacenó a -80 º C hasta su uso. </li><li> Antes de cultivo, la mezcla del líquido sinovial con el medio de diferenciación de los condrocitos (MDL, Lonza) en diferentes proporciones en volumen, con una concentración constante de ácido ascórbico de 100 mm y 9 mm de CaCl2 (esta cantidad mínima de CaCl2 es asegurar la integridad del alginato cuentas). </li><li> Mantener la placa en una plataforma de mecerse en una incubadora de 37 ° C (frecuencia de oscilación: alrededor de 75 veces / min) para ayudar a la distribución de nutrientes dentro de las perlas de alginato, y para reducir la aglutinación de las bolas. Esto es especialmente importante a medida que la cultura los condrocitos durante un período prolongado de tiempo (hasta 4 semanas). Nota: Si bien el crecimiento de condrocitos y medio de diferenciación (Lonza) contienen dos antibióticos de uso común gentamicina y anfotericina, no complementar los antibióticos cuando se utilizan diferentes porcentajes de líquido sinovial para el cultivo de condrocitos. No se observó contaminación nunca. </li><li> Cambiar mezclas de medios cada 2-3 días. La viscosidad del líquido sinovial ha sido un obstáculo importante en el cultivo de condrocitos encapsulados en perlas de alginato en cultivos a largo plazo. Nos pareció que era más eficaz para diluir el líquido sinovial-medio suplementado con 102 mm de CaCl 2 (50% V / V), que también fortalece la integridad de las perlas de alginato. De esta manera, el medio poco a poco se puede quitar con una pipeta, sin dañar los granos. </li></ol> 4. HAC cosecha en perlas de alginato para el análisis de expresión génica Especial cuidado debe tenerse cuando se cosecha el HAC de las perlas de alginato para el análisis de expresión génica. <ol><li> Lavar las perlas de alginato tres times en 102 mM CaCl 2 durante 5 minutos cada vez. </li><li> Recuperar los condrocitos mediante la adición de 55 mM NaCitrate a un volumen 4.5 veces mayor que la de las cuentas. Es importante que las perlas de alginato están totalmente inmersos en la solución NaCitrate. Agitar o roca durante 20-30 min. </li><li> Centrifugar las células y se desecha el sobrenadante. </li><li> De análisis RT-PCR, resuspender el botón celular en tampón de lisis celular (Qiagen kit de aislamiento de ARN), y proceder a la purificación de ARN. </li></ol> 5. Fix HAC en perlas de alginato para el análisis histológico Especial cuidado hay que tener para cosechar el HAC de las cuentas en 3D para su análisis histológico. <ol><li> Lavar las perlas de alginato 3 veces en 102 mM CaCl 2 durante 5 minutos cada vez. Para lavar completamente el líquido sinovial viscoso, nos pareció que era más eficaz para lavar con el medio de diferenciación de los condrocitos (Lonza) durante la noche, meciéndose en una incubadora de 37 ° C (frecuencia de oscilación: 75 horas / min). El medio de crecimiento de los condrocitos se utilizó para asegurar la supervivencia de los condrocitos en este lavado prolongado y para permitir la máxima penetración del agente de fijación. </li><li> Arreglar las cuentas en el 70% de etanol durante la noche, y proceder con el análisis histológico. Para llevar a cabo DAPI, incubar perlas de alginato con DAPI solución (500ng/ml) durante 1 hora en suave balanceo, y lavar 3 veces con 102 mm de CaCl2 durante 15 minutos cada vez. Dapi imágenes se pueden observar bajo un microscopio de fluorescencia. Las cuentas también se puede seccionar de Alcian azul y tinción H &amp; E. </li></ol> 6. Resultados representante Nuestro método de cultivo de condrocitos humanos en 3D en un alto porcentaje de líquido sinovial se muestra en el diagrama esquemático se muestra en la Fig. 1. Después de los condrocitos humanos se concentraron en perlas de alginato, se les permitió crecer en un medio suplementado con diferentes proporciones de líquido sinovial. Debido a la viscosidad del líquido sinovial, que es esencial paracondrocitos cultivo bajo condiciones de balanceo constante para evitar la aglutinación de las construcciones de los cartílagos y para asegurar una distribución uniforme de los nutrientes. También es esencial para lavar las perlas de alginato ampliamente antes de recuperar los condrocitos, de modo que la fijación o tampón de lisis puede penetrar en las cuentas (Fig. 1). Campo claro (BF) las imágenes de los condrocitos en las perlas de alginato se muestra en la Fig. 2. DAPI se realizó para confirmar la distribución usniform de las células dentro de las perlas (Fig. 2). Al final del período de cultivo de 21 días, análisis de expresión génica se realizó mediante QRT-PCR. La referencia del gen GAPDH se utilizó para la normalización de todos los informes de terminación, ya que se determinó que era uno de los genes de referencia más fiable para el análisis de PCR cuantitativa en los condrocitos 32. Un ejemplo se muestra en la fig. 3, donde se analizaron los resultados de los condrocitos articulares humanos cultivados en el líquido sinovial agrupados de seis pacientes con osteoartritis. De acuerdo con el hecho de que los condrocitos deLonza se han extendido en las culturas en 2D, lo que conduciría inevitablemente a la des-diferenciación de 33 años, encontramos que el día 0 condrocitos expresan niveles mínimos de genes de la matriz del cartílago (Fig. 3). Cultivo de condrocitos en 3D con el medio de diferenciación de los condrocitos (Lonza) o el medio suplementado con el líquido sinovial de manera significativa el aumento de expresión del gen del colágeno de cartílago, aggrecan y MMP13, lo que indica condrocitos volver a la diferenciación por el día 21 (Fig. 3) 34. Aumentar el porcentaje de líquido sinovial en los medios de comunicación dio lugar a niveles comparables de la matriz del cartílago marcadores II de colágeno y la expresión de ARNm aggrecan (Fig.3a y 3B). Además, los condrocitos cultivados en el líquido sinovial, incluso el 100% mostraron una disminución en la expresión de ARNm de enzima que degrada el cartílago MMP13 en comparación con los cultivados en el medio solo (Fig.3C). Curiosamente, el nivel de expresión de la caspasa muerte celular indicador 3 disminuyó gradualmente con el aumento de ratios de líquido sinovial, lo que sugiere que scultivo de líquido ynovial ha llevado a niveles de disminución de la apoptosis (Fig. 3D). Por lo tanto, nuestros resultados muestran que el cultivo de condrocitos humanos articular en los altos niveles de líquido sinovial en un entorno 3D es una tecnología viable. Tablas y Figuras <img alt="Figura 1." src="/files/ftp_upload/3587/3587fig1.jpg" /> Figura 1. Diagrama esquemático del método de los condrocitos articulares humanos en la cultura un alto porcentaje de líquido sinovial en perlas de alginato 3D. En primer lugar, los condrocitos y la solución de alginato se mezclan. Cuando se aplica gota a gota a la solución de CaCl2, los condrocitos se encuentran inmovilizados en las cuentas de hidrogel reticulado alginato de calcio. Estas construcciones 3D luego se cultivaron en el medio de diferenciación de condrocitos (Lonza) con proporciones variables de líquido sinovial humano. Después de 21 días de cultivo bajo condiciones de balanceo, perlas de alginato que contienen las células se lavan ampliamente después de que las células se recuperan para el genanálisis de la expresión. <img alt="Figura 2." src="/files/ftp_upload/3587/3587fig2.jpg" /> Figura 2. De campo claro (BF) y las imágenes Dapi de los condrocitos articulares humanos culturas encapsulado con 0%, 30%, 50%, 70% y 100% del líquido sinovial. Condrocitos fueron de forma esférica en todas las condiciones de cultivo. Las imágenes de campo claro (BF) y DAPI se superponen para confirmar la ubicación y distribución de los condrocitos. Recuadros, imágenes ampliadas. Puntas de flecha, colocalización de los condrocitos en BF y las imágenes Dapi manchas. <img alt="Figura 3." src="/files/ftp_upload/3587/3587fig3.jpg" /> Figura 3. QRT-PCR análisis de encapsulado condrocitos articulares humanos en el día 0 (D0) una después de 21 días de cultivo (D21) en un medio suplementado con diferentes proporciones de líquido sinovial (SF) (0%, 30%, 50%, 70 % y 100%). Resultados de cuatro muestras independientes se muestran aquí. GAPDH se utilizó como referencia interna para todos los ITP. <strong&gt; A. Colágeno II expresión del ARNm. B. Aggrecan la expresión del ARNm. C. MMP13 expresión del ARNm. D. Caspasa 3 mRNA expresión. La significación estadística se evaluó por el Día 0 y Día de las muestras de 21 muestras de 0% y 100% del líquido sinovial (SF) el cultivo, el uso de software INSTAT. * Indica P &lt;0,05.

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+synovial+fluid+and+synovial+fluid+cells+on+chondrocyte+metabolism+in+short+term+tissue+culture.">Effects of synovial fluid and synovial fluid cells on chondrocyte metabolism in short term tissue culture.</a> J. Rheumatol. 15, 210-216 (1988).</li><li>Beekhuizen, M., Bastiaansen-Jenniskens, Y. M., Koevoet, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+tissue+inhibits+proteoglycan+production+in+human+osteoarthritic+cartilage;+Establishment+and+characterisation+of+a+long-term+coculture.">Osteoarthritic synovial tissue inhibits proteoglycan production in human osteoarthritic cartilage; Establishment and characterisation of a long-term coculture.</a> Arthritis Rheum. , (2011).</li><li>Rodrigo, J. J., Steadman, J. R., Syftestad, G., Benton, H., Silliman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+human+knee+synovial+fluid+on+chondrogenesis+in+vitro.">Effects of human knee synovial fluid on chondrogenesis in vitro.</a> Am. J. Knee. Surg. 8, 124-129 (1995).</li><li>van den Hoogen, B. M., van de Lest, C. H., van Weeren, P. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loading-induced+changes+in+synovial+fluid+affect+cartilage+metabolism.">Loading-induced changes in synovial fluid affect cartilage metabolism.</a> Br. J. Rheumatol. 37, 671-676 (1998).</li><li>Webb, G. R., Westacott, C. I., Elson, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Osteoarthritic+synovial+fluid+and+synovium+supernatants+up-regulate+tumor+necrosis+factor+receptors+on+human+articular+chondrocytes.">Osteoarthritic synovial fluid and synovium supernatants up-regulate tumor necrosis factor receptors on human articular chondrocytes.</a> Osteoarthritis Cartilage. 6, 167-176 (1998).</li><li>Kook, S. H., Son, Y. O., Lee, K. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+affects+positively+the+proliferation+of+bovine+satellite+cells+and+their+myogenic+differentiation+through+up-regulation+of+MyoD.">Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD.</a> Cell. Biol. Int. 32, 871-878 (2008).</li><li>Knobloch, T. J., Madhavan, S., Nam, J., Agarwal, S., Agarwal, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+of+chondrocytic+gene+expression+by+biomechanical+signals.">Regulation of chondrocytic gene expression by biomechanical signals.</a> Crit. Rev. Eukaryot. Gene. Expr. 18, 139-150 (2008).</li><li>Guo, J. F., Jourdian, G. W., MacCallum, D. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culture+and+growth+characteristics+of+chondrocytes+encapsulated+in+alginate+beads.">Culture and growth characteristics of chondrocytes encapsulated in alginate beads.</a> Connect. Tissue. Res. 19, 277-297 (1989).</li><li>Toegel, S., Huang, W., Piana, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selection+of+reliable+reference+genes+for+qPCR+studies+on+chondroprotective+action.">Selection of reliable reference genes for qPCR studies on chondroprotective action.</a> BMC. Mol. Biol. 8, 13-13 (2007).</li><li>Lin, Z., Fitzgerald, J. B., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiles+of+human+chondrocytes+during+passaged+monolayer+cultivation.">Gene expression profiles of human chondrocytes during passaged monolayer cultivation.</a> J. Orthop. Res. 26, 1230-1237 (2008).</li><li>Goessler, U. R., Bieback, K., Bugert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chondrocytes+differentially+express+matrix+modulators+during+in+vitro+expansion+for+tissue+engineering.">Human chondrocytes differentially express matrix modulators during in vitro expansion for tissue engineering.</a> Int. J. Mol. Med. 16, 509-515 (2005).</li><li>Anat, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> 121, 107-118 (1976).</li></ol>

No tenemos nada que revelar.

En este informe, hemos desarrollado un método que permite el cultivo de condrocitos articulares humanos en un entorno 3D en un medio que contiene altas concentraciones de líquido sinovial humano. El líquido sinovial es uno de los principales componentes que constituyen el entorno natural en la cavidad de la articulación, donde residen los condrocitos articulares. Sin embargo, la viscosidad del líquido sinovial ha sido un gran desafío para tres dimensiones a largo plazo el cultivo de condrocitos. Para superar el re...

Tabla de reactivos y equipos específicos: <table border="1"><tr><th> Nombre del reactivo </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios </th></tr><tr><td> Alginato (sal sódica del ácido algínico) </td><td> Sigma </td><td> A2158-250G </td><td> 2.4% de solución de almacenado a 40 ° C </td></tr><tr><td> Cloruro de calcio dihidrato, Granular </td><td> JT Baker </td><td> A19339 </td><td></td></tr><tr><td width="239" rowspan="2" > Los medios de comunicación condrogénica crecimiento </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3156 (base de los medios de comunicación) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4409 (con suplemento) </td></tr><tr><td width="239" rowspan="2" > Los medios de comunicación condrogénica diferenciación </td><td width="254" rowspan="2" > Lonza </td><td width="182" > CC-3226 (base de los medios de comunicación) </td><td width="141" rowspan="2" ></td></tr><tr><td width="182" > CC-4408 (con suplemento) </td></tr><tr><td> Condrocitos articulares humanos </td><td> Lonza </td><td > CC-2550 </td><td></td></tr><tr><td> DAPI (4 &#39;,6-diamidino-2-phenylindole diclorhidrato) </td><td> Sigma-Aldrich </td><td > D9542 </td><td></td></tr><tr><td> RNeasy mini kit (para la extracción de ARN) </td><td> Qiagen </td><td > 74104 </td><td></td></tr><tr><td> PCR reactivos: SYBR-verde </td><td> Quanta </td><td > 95053-500 </td><td></td></tr><tr><td> 12 ml jeringa </td><td> Tyco-Kendall-Monoject </td><td > 512852 </td><td></td></tr><tr><td> 22 Gague aguja hipodérmica </td><td> Tyco-Kendall-Monoject </td><td > 8881 </td><td></td></tr><tr><td> Microscopio </td><td> Olimpo </td><td > IX71 </td><td></td></tr><tr><td> Plataforma oscilante </td><td> Thermolyne Thermoscientific </td><td > Vari-mix </td><td></td></tr><tr><td></td><td></td><td ></td><td></td></tr><tr><td> Cebadores secuencias </td><td></td><td ></td><td></td></tr><tr><td> Colágeno IIa-forward </td><td> 5&#39;-TTC ATC CCA CCC TCT AGT CAC-3 &#39; </td><td ></td><td></td></tr><tr><td> Colágeno II a invertir </td><td> 5&#39;-CCTCTGCCTTGACCCGAA-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-forward </td><td> 5&#39;-GCC TGT CTT CTT CAC ACA ACT GAC-3 &#39; </td><td ></td><td></td></tr><tr><td> MMP13-reverse </td><td> 5&#39;-GAG AGC AGA CTT TGA GTC TCA GCC-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspasa-3 hacia adelante </td><td> 5&#39;-TTC TTA TCA AGG CCT GCC GTG GTA-3 &#39; </td><td ></td><td></td></tr><tr><td> Caspasa-3 inversa </td><td> 5&#39;-TGG ATG AAC CAG CCA GAG TCC TTT -3 &#39; </td><td ></td><td></td></tr></table>

a 3d system for culturing human articular chondrocytes in synovial fluid

Destrucción del cartílago es una característica central patológicas de la artrosis, la principal causa de discapacidad en los EE.UU.. Cartílago en el adulto no se regenera de manera muy eficiente en vivo, y como consecuencia, conduce a la osteoartritis irreversible pérdida de cartílago y se acompaña de dolor crónico y 1,2 inmovilidad. Ingeniería tisular del cartílago ofrece un potencial prometedor para regenerar y restaurar la función del tejido. Esta tecnología implica normalmente la siembra de los condrocitos en andamios natural o sintético y el cultivo de las 3D da como resultado la construcción en un medio equilibrado en un período de tiempo con el objetivo de la ingeniería bioquímica y un tejido maduro biomecánica que pueden ser trasplantados en un sitio de defecto en vivo 3-6 . Lograr una condición óptima para el crecimiento de los condrocitos y la deposición de la matriz es esencial para el éxito de la ingeniería de tejido cartilaginoso. En la cavidad de la articulación del cartílago nativo, en el árticocular la superficie del hueso está bañado en el líquido sinovial. Este líquido claro y viscoso, proporciona nutrientes al cartílago articular avascular y contiene factores de crecimiento, citoquinas y enzimas que son importantes para el metabolismo de los condrocitos 7,8. Además, el líquido sinovial facilita el movimiento de baja fricción entre las superficies cartilaginosas, principalmente a través de secretar dos componentes clave, ácido hialurónico y lubricina 9 10. En contraste, el cartílago de la ingeniería tisular es más a menudo cultivadas en medios artificiales. Si bien estos medios de comunicación es probable ofrecer condiciones más definido para el estudio del metabolismo de condrocitos, el líquido sinovial refleja con más precisión el entorno natural de que los condrocitos articulares residen pulg De hecho, el líquido sinovial tiene la ventaja de ser fáciles de obtener y almacenar, ya menudo puede ser renueva constantemente por el cuerpo. Varios grupos han complementado el medio de cultivo de líquido sinovial en el crecimiento humano, conejo, bovino, y el perro chondrocytes, pero la mayoría utilizan bajos niveles de líquido sinovial (por debajo del 20%) 11-25. Mientras que los condrocitos de pollo, caballo y humanos han sido cultivadas en el medio con mayor porcentaje de líquido sinovial, estos sistemas de cultivo de dos dimensiones 26-28. A continuación les presentamos nuestro método de cultivo de condrocitos articulares humanos en un sistema 3D con un alto porcentaje de líquido sinovial (hasta el 100%) durante un período de 21 días. Al hacerlo, hemos superado un gran obstáculo presentado por la alta viscosidad del líquido sinovial. Este sistema ofrece la posibilidad de estudiar los condrocitos humanos en el líquido sinovial en un entorno 3D, que puede combinarse con otros dos factores importantes (la tensión de oxígeno y la carga mecánica) 29,30 que constituyen el entorno natural de cartílago para imitar el medio natural para cartílago de crecimiento. Además, este sistema también puede ser utilizado para el ensayo de la actividad de líquido sinovial en los condrocitos y proporcionar una plataforma para el desarrollo deregeneración del cartílago tecnologías y opciones terapéuticas para la artritis.

Watch this Scientific Journal Video about A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid at JoVE.com

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Un sistema 3D de los condrocitos articulares humanos en cultivo de altos niveles de líquido sinovial se describe. El líquido sinovial refleja el microambiente más naturales para el cartílago articular, y puede ser fácilmente obtenido y almacenado. Así, este sistema puede ser utilizado para el estudio de la regeneración del cartílago y de terapias de detección para el tratamiento de la artritis.

Un sistema 3D para los condrocitos articulares humanos cultivo de líquido sinovial

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

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Research

JoVE Journal

Biology

20.7K vistas.  Tufts University School of Medicine. Un sistema 3D de los condrocitos articulares humanos en cultivo de altos niveles de líquido sinovial se describe. El líquido sinovial refleja el microambiente más naturales para el cartílago articular, y puede ser fácilmente obtenido y almacenado. Así, este sistema puede ser utilizado para el estudio de la regeneración del cartílago y de terapias de detección para el tratamiento de la artritis.

Video: A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

A System for Culturing Iris Pigment Epithelial Cells to Study Lens Regeneration in Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, heart, the jaw 1. Specifically, newts are unique for its lens regeneration capability. Upon lens removal, IPE cells of the dorsal iris transdifferentiate to lens cells and eventually form a new lens in about a month 2,3. This property of regeneration is never exhibited by the ventral iris cells. The regeneration potential of the iris cells can be studied by making transplants of the in vitro cultured IPE cells. For the culture, the dorsal and ventral iris cells are first isolated from the eye and cultured separately for a time period of 2 weeks (Figure 1). These cultured cells are reaggregated and implanted back to the newt eye. Past studies have shown that the dorsal reaggregate maintains its lens forming capacity whereas the ventral aggregate does not form a lens, recapitulating, thus the in vivo process (Figure 2) 4,5. This system of determining regeneration potential of dorsal and ventral iris cells is very useful in studying the role of genes and proteins involved in lens regeneration.

In newt, the lens regenerates always from the dorsal iris by transdifferentiation of the iris pigmented epithelial cells (IPEs). Here we describe a procedure to culture dorsal and ventral newt IPE cells and their implantation to the newt eye. The implanted cells are then studied by tissue sectioning and immunohistochemistry.

Un sistema para el cultivo de células epiteliales del pigmento del iris para el Estudio de Regeneración del objetivo de Newt

Salamanders like newt and axolotl possess the ability to regenerate many of its lost body parts such as limbs, the tail with spinal cord, eye, brain, ...

Investigación

Biología

Efficient Derivation of Human Cardiac Precursors and Cardiomyocytes from Pluripotent Human Embryonic Stem Cells with Small Molecule Induction

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based transplantation or by cardiac tissue engineering1-3. Cardiomyocytes become terminally-differentiated soon after birth and lose their ability to proliferate. There is no evidence that stem/progenitor cells derived from other sources, such as the bone marrow or the cord blood, are able to give rise to the contractile heart muscle cells following transplantation into the heart1-3. The need to regenerate or repair the damaged heart muscle has not been met by adult stem cell therapy, either endogenous or via cell delivery1-3. The genetically stable human embryonic stem cells (hESCs) have unlimited expansion ability and unrestricted plasticity, proffering a pluripotent reservoir for in vitro derivation of large supplies of human somatic cells that are restricted to the lineage in need of repair and regeneration4,5. Due to the prevalence of cardiovascular disease worldwide and acute shortage of donor organs, there is intense interest in developing hESC-based therapies as an alternative approach. However, how to channel the wide differentiation potential of pluripotent hESCs efficiently and predictably to a desired phenotype has been a major challenge for both developmental study and clinical translation. Conventional approaches rely on multi-lineage inclination of pluripotent cells through spontaneous germ layer differentiation, resulting in inefficient and uncontrollable lineage-commitment that is often followed by phenotypic heterogeneity and instability, hence, a high risk of tumorigenicity6-8 (see a schematic in Fig. 1A). In addition, undefined foreign/animal biological supplements and/or feeders that have typically been used for the isolation, expansion, and differentiation of hESCs may make direct use of such cell-specialized grafts in patients problematic9-11. To overcome these obstacles, we have resolved the elements of a defined culture system necessary and sufficient for sustaining the epiblast pluripotence of hESCs, serving as a platform for de novo derivation of clinically-suitable hESCs and effectively directing such hESCs uniformly towards clinically-relevant lineages by small molecules12 (see a schematic in Fig. 1B). After screening a variety of small molecules and growth factors, we found that such defined conditions rendered nicotinamide (NAM) sufficient to induce the specification of cardiomesoderm direct from pluripotent hESCs that further progressed to cardioblasts that generated human beating cardiomyocytes with high efficiency (Fig. 2). We defined conditions for induction of cardioblasts direct from pluripotent hESCs without an intervening multi-lineage embryoid body stage, enabling well-controlled efficient derivation of a large supply of human cardiac cells across the spectrum of developmental stages for cell-based therapeutics.

We have established a protocol for induction of cardioblasts direct from pluripotent human embryonic stem cells maintained under defined conditions with small molecules, which enables derivation of a large supply of human cardiac progenitors and functional cardiomyocytes for cardiovascular repair.

Derivación eficiente de los Derechos Humanos precursores cardiacos y cardiomiocitos de pluripotentes células madre embrionarias humanas mediante la inducción de moléculas pequeñas

To date, the lack of a suitable human cardiac cell source has been the major setback in regenerating the human myocardium, either by cell-based ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un sistema para la<em&gt; Ex vivo</em&gt; Cultivo de páncreas embrionario

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer's Vesicle

Internal organs such as the heart, brain, and gut develop left-right (LR) asymmetries that are critical for their normal functions1. Motile cilia are involved in establishing LR asymmetry in vertebrate embryos, including mouse, frog, and zebrafish2-6. These 'LR cilia' generate asymmetric fluid flow that is necessary to trigger a conserved asymmetric Nodal (TGF-&beta; superfamily) signaling cascade in the left lateral plate mesoderm, which is thought to provide LR patterning information for developing organs7. Thus, to understand mechanisms underlying LR patterning, it is essential to identify genes that regulate the organization of LR ciliated cells, the motility and length of LR cilia and their ability to generate robust asymmetric flow.
In the zebrafish embryo, LR cilia are located in Kupffer's vesicle (KV)2,4,5. KV is comprised of a single layer of monociliated epithelial cells that enclose a fluid-filled lumen. Fate mapping has shown that KV is derived from a group of ~20-30 cells known as dorsal forerunner cells (DFCs) that migrate at the dorsal blastoderm margin during epiboly stages8,9. During early somite stages, DFCs cluster and differentiate into ciliated epithelial cells to form KV in the tailbud of the embryo10,11. The ability to identify and track DFCs&mdash;in combination with optical transparency and rapid development of the zebrafish embryo&mdash;make zebrafish KV an excellent model system to study LR ciliated cells.
Interestingly, progenitors of the DFC/KV cell lineage retain cytoplasmic bridges between the yolk cell up to 4 hr post-fertilization (hpf), whereas cytoplasmic bridges between the yolk cell and other embryonic cells close after 2 hpf8. Taking advantage of these cytoplasmic bridges, we developed a stage-specific injection strategy to deliver morpholino oligonucleotides (MO) exclusively to DFCs and knockdown the function of a targeted gene in these cells12. This technique creates chimeric embryos in which gene function is knocked down in the DFC/KV lineage developing in the context of a wild-type embryo. To analyze asymmetric fluid flow in KV, we inject fluorescent microbeads into the KV lumen and record bead movement using videomicroscopy2. Fluid flow is easily visualized and can be quantified by tracking bead displacement over time.
Here, using the stage-specific DFC-targeted gene knockdown technique and injection of fluorescent microbeads into KV to visualize flow, we present a protocol that provides an effective approach to characterize the role of a particular gene during KV development and function.

Analysis of Gene Function and Visualization of Cilia-Generated Fluid Flow in Kupffer&#039;s Vesicle

Cilia-generated fluid flow in Kupffer&#x2019;s Vesicle (KV) controls left-right patterning of the zebrafish embryo. Here, we describe a technique to modulate gene function specifically in KV cells. In addition, we show how to deliver fluorescent beads into KV to visualize fluid flow.

El análisis de la función génica y visualización de los cilios-generó un flujo de líquido en la vesícula Kupffer

Internal organs such as the  heart, brain, and gut develop left-right (LR) asymmetries that are critical for  their normal functions1. Motile cilia are ...

Culturing of Human Nasal Epithelial Cells at the Air Liquid Interface

In vitro models using human primary epithelial cells are essential in understanding key functions of the respiratory epithelium in the context of microbial infections or inhaled agents. Direct comparisons of cells obtained from diseased populations allow us to characterize different phenotypes and dissect the underlying mechanisms mediating changes in epithelial cell function. Culturing epithelial cells from the human tracheobronchial region has been well documented, but is limited by the availability of human lung tissue or invasiveness associated with obtaining the bronchial brushes biopsies. Nasal epithelial cells are obtained through much less invasive superficial nasal scrape biopsies and subjects can be biopsied multiple times with no significant side effects. Additionally, the nose is the entry point to the respiratory system and therefore one of the first sites to be exposed to any kind of air-borne stressor, such as microbial agents, pollutants, or allergens. 
Briefly, nasal epithelial cells obtained from human volunteers are expanded on coated tissue culture plates, and then transferred onto cell culture inserts. Upon reaching confluency, cells continue to be cultured at the air-liquid interface (ALI), for several weeks, which creates more physiologically relevant conditions. The ALI culture condition uses defined media leading to a differentiated epithelium that exhibits morphological and functional characteristics similar to the human nasal epithelium, with both ciliated and mucus producing cells. Tissue culture inserts with differentiated nasal epithelial cells can be manipulated in a variety of ways depending on the research questions (treatment with pharmacological agents, transduction with lentiviral vectors, exposure to gases, or infection with microbial agents) and analyzed for numerous different endpoints ranging from cellular and molecular pathways, functional changes, morphology, etc. 
In vitro models of differentiated human nasal epithelial cells will enable investigators to address novel and important research questions by using organotypic experimental models that largely mimic the nasal epithelium in vivo.

Nasal epithelial cells, obtained through superficial scrape biopsy of human volunteers, are expanded and transferred onto tissue culture inserts. Upon reaching confluency, cells are grown at air liquid interface, yielding cultures of ciliated and non-ciliated cells. Differentiated nasal epithelial cell cultures provide viable experimental models for studying the respiratory mucosa.

El cultivo de células humanas epiteliales nasales en la interfase aire líquido

In vitro models using human primary  epithelial cells are essential in understanding key functions of the respiratory  epithelium in the context of ...

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the function and regulation of protein folding was studied in vitro, in isolated tissue culture cells and in unicellular organisms. Recent studies have uncovered links between protein homeostasis (proteostasis), metabolism, development, aging, and temperature-sensing. These findings have led to the development of new tools for monitoring protein folding in the model metazoan organism Caenorhabditis elegans. In our laboratory, we combine behavioral assays, imaging and biochemical approaches using temperature-sensitive or naturally occurring metastable proteins as sensors of the folding environment to monitor protein misfolding. Behavioral assays that are associated with the misfolding of a specific protein provide a simple and powerful readout for protein folding, allowing for the fast screening of genes and conditions that modulate folding. Likewise, such misfolding can be associated with protein mislocalization in the cell. Monitoring protein localization can, therefore, highlight changes in cellular folding capacity occurring in different tissues, at various stages of development and in the face of changing conditions. Finally, using biochemical tools ex vivo, we can directly monitor protein stability and conformation. Thus, by combining behavioral assays, imaging and biochemical techniques, we are able to monitor protein misfolding at the resolution of the organism, the cell, and the protein, respectively.

Using Caenorhabditis elegans as a Model System to Study Protein Homeostasis in a Multicellular Organism

To study the relationship between protein homeostasis, stress and aging, we monitored changes in protein folding by following protein dysfunction, protein localization in the cell and protein stability at the organismal, cellular and protein levels, using the genetically tractable metazoan Caenorhabditis elegans as a model system.

The folding and assembly of proteins is essential for protein function, the long-term health of the cell, and longevity of the organism. Historically, the ...

Gap Junctional Intercellular Communication: A Functional Biomarker to Assess Adverse Effects of Toxicants and Toxins, and Health Benefits of Natural Products

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction channels, which is a major intercellular process by which tissue homeostasis is maintained. Interruption of gap junctional intercellular communication (GJIC) by toxicants, toxins, drugs, etc. has been linked to numerous adverse health effects. Many genetic-based human diseases have been linked to mutations in gap junction genes. The SL-DT technique is a simple functional assay for the simultaneous assessment of GJIC in a large population of cells. The assay involves pre-loading cells with a fluorescent dye by briefly perturbing the cell membrane with a scalpel blade through a population of cells. The fluorescent dye is then allowed to traverse through gap junction channels to neighboring cells for a designated time. The assay is then terminated by the addition of formalin to the cells. The spread of the fluorescent dye through a population of cells is assessed with an epifluorescence microscope and the images are analyzed with any number of morphometric software packages that are available, including free software packages found on the public domain. This assay has also been adapted for in vivo studies using tissue slices from various organs from treated animals. Overall, the SL-DT assay can serve a broad range of in vitro pharmacological and toxicological needs, and can be potentially adapted for high throughput set-up systems with automated fluorescence microscopy imaging and analysis to elucidate more samples in a shorter time.

This protocol describes a scalpel loading-fluorescent dye transfer technique that measures intercellular communication through gap junction channels. Gap junctional intercellular communication is a major cellular process by which tissue homeostasis is maintained and disruption of this cell signaling has adverse health effects.

Brecha ocasiones intercelular de comunicación: un biomarcador funcional para evaluar los efectos adversos de las sustancias tóxicas y toxinas, y de Salud Beneficios de Productos Naturales

This protocol describes a scalpel loading-fluorescent dye transfer (SL-DT) technique that measures intercellular communication through gap junction ...

The Assembly and Application of 'Shear Rings': A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as well as pathological changes in endothelial phenotype and gene expression. In particular, maladaptive effects of periodic, unidirectional flow induced shear stress can trigger a variety of effects on several vascular cell types, particularly endothelial cells. While by now endothelial cells from diverse anatomic origins have been cultured, in-depth analyses of their responses to fluid shear have been hampered by the relative complexity of shear models (e.g., parallel plate flow chamber, cone and plate flow model). While these all represent excellent approaches, such models are technically complicated and suffer from drawbacks including relatively lengthy and complex setup time, low surface areas, requirements for pumps and pressurization often requiring sealants and gaskets, creating challenges to both maintenance of sterility and an inability to run multiple experiments. However, if higher throughput models of flow and shear were available, greater progress on vascular endothelial shear responses, particularly periodic shear research at the molecular level, might be more rapidly advanced. Here, we describe the construction and use of shear rings: a novel, simple-to-assemble, and inexpensive tissue culture model with a relatively large surface area that easily allows for a high number of experimental replicates in unidirectional, periodic shear stress studies on endothelial cells.

The Assembly and Application of &#039;Shear Rings&#039;: A Novel Endothelial Model for Orbital, Unidirectional and Periodic Fluid Flow and Shear Stress

Different levels and patterns of fluid shear are known to modulate endothelial gene expression, phenotype and susceptibility to disease. We discuss the assembly and use of 'shear rings': a model that produces unidirectional, periodic shear stress patterns. Shear rings are simple to assemble, economical and can produce high cell yields.

La Asamblea y la Aplicación de &#39;Shear Anillos&#39;: A Novel endotelial Modelo de Orbital, unidireccional y periódica flujo de fluido y la tensión de cizallamiento

Deviations from normal levels and patterns of vascular fluid shear play important roles in vascular physiology and pathophysiology by inducing adaptive as ...

Magnetic-Activated Cell Sorting Strategies to Isolate and Purify Synovial Fluid-Derived Mesenchymal Stem Cells from a Rabbit Model

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for cartilage tissue engineering. MSCs from synovial fluid (SF-MSCs) have been considered promising candidates for articular regeneration, and their potential therapeutic benefit has made them an important research topic of late. SF-MSCs from the knee cavity of the New Zealand white rabbit can be employed as an optimized translational model to assess human regenerative medicine. By means of CD90-based magnetic activated cell sorting (MACS) technologies, this protocol successfully obtains rabbit SF-MSCs (rbSF-MSCs) from this rabbit model and further fully demonstrates the MSC phenotype of these cells by inducing them to differentiate to osteoblasts, adipocytes, and chondrocytes. Therefore, this approach can be applied in cell biology research and tissue engineering using simple equipment and procedures.

This article presents a simple and economic protocol for the straightforward isolation and purification of mesenchymal stem cells from New Zealand white rabbit synovial fluid.

Célula activada por el magnético clasificación estrategias para aislar y purificar sinovial líquido mesenquimales células madre derivadas de un modelo de conejo

Mesenchymal stem cells (MSCs) are the main cell source for cell-based therapy. MSCs from articular cavity synovial fluid could potentially be used for ...

3D Culturing of Organoids from the Intestinal Villi Epithelium Undergoing Dedifferentiation

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is compartmentalized into crypts and villi: the stem and proliferating cells are confined to the crypts, whereas the villi epithelium contains only differentiated cells. Hence, the normal intestinal crypts, but not the villi, can give rise to organoids in 3D cultures. The procedure described here is applicable only to villus epithelium undergoing dedifferentiation leading to stemness. The method described uses the Smad4-loss-of-function:&#946;-catenin gain-of-function (Smad4KO:&#946;-cateninGOF) conditional mutant mouse. The mutation causes the intestinal villi to dedifferentiate and generate stem cells in the villi. Intestinal villi undergoing dedifferentiation are scraped off the intestine using glass slides, placed in a 70 &#181;m strainer and washed several times to filter out any loose cells or crypts prior to plating in BME-R1 matrix to determine their organoid-forming potential. Two main criteria were used to ensure that the resulting organoids were developed from the dedifferentiating villus compartment and not from the crypts: 1) microscopically evaluating&#160;the isolated villi to ensure absence of any tethered crypts, both before and after plating in the 3D matrix, and 2) monitoring the time course of organoid development from the villi. Organoid initiation from the villi occurs only two to five days after plating and appears irregularly shaped, whereas the crypt-derived organoids from the same intestinal epithelium are apparent within sixteen hours of plating and appear spherical. The limitation of the method, however, is that the number of organoids formed, and the time required for organoid initiation from the villi vary depending on the degree of dedifferentiation. Hence, depending upon the specificity of the mutation or the insult causing the dedifferentiation, the optimal stage at which villi can be harvested to assay their organoid forming potential, must be determined empirically.

The procedure describes isolation of the villi from the mouse intestinal epithelium undergoing dedifferentiation to determine their organoid forming potential.

Cultivo 3D de organoides del epitelio de las vellosidades intestinales sometidos a desdiferenciación

Clonogenicity of organoids from the intestinal epithelium is attributed to the presence of stem cells therein. The mouse small intestinal epithelium is ...