Name: engineered 3d silk-collagen-based model of polarized neural tissue
Uploaded: 2015-10-23T14:00:00
Duration: 6 min 17 s
Description: Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

We thank the laboratory of Dr. Stephen Moss for providing embryonic rat brain tissues. M.D.T. designed the original protocol. This work was funded by National Institutes of Health P41 Tissue Engineering Resource Center Grant EB002520. K.C. was supported with Postdoctoral Fellowship from German Research Foundation (DFG) (CH 1400/2-1).

El protocolo de aislamiento de tejido cerebral fue aprobado por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Tufts y cumple con los Institutos Nacionales de Salud de Guía para el Cuidado y Uso de Animales de Laboratorio (Institucional Cuidado de Animales y el empleo Comisión B2011-45). 1. Preparación de la Seda Andamios <ol><li> Preparación de la solución de seda de los capullos Bombyx mori como se describió previamente 19,20. <ol><li> Corte cada capullo en 8 partes iguales utilizando tijeras. Utilice unos 11 capullos de 5 g de capullos fragmentados. (15 minutos) </li><li> Prepare 2 L de M Na 2 CO 3 solución de 0,02 y ponerla a hervir utilizando una placa caliente. (15 minutos) </li><li> Pesar 5 g de capullos fragmentadas y hervirlas en Na 2 CO 3 solución durante 30 minutos. Revuelva la seda hirviendo cada par de minutos con una espátula. Este paso, también llamada de-engomado, purifica fibroína de seda de proteínas hidrófilas, sericins. (30 minutos) </li><li&gt; Escurra la fibroína a mano y enjuague en agua destilada al menos tres veces para lavar cualquier sericina restante y productos químicos. (5 minutos) </li><li> Coloque la fibroína húmedo en una placa de Petri y se seca el extracto fibroína en la campana de flujo O / N. </li><li> Al día siguiente, pesa la masa fibroína seco y colocar la fibroína en un vaso de vidrio. (15 minutos) </li><li> Con el fin de disolver la fibroína en solución 9,3 M LiBr, calcular el volumen requerido (en ml) de LiBr multiplicando la masa de fibroína seca por 4. Verter lentamente solución de LiBr sobre la fibroína de seda y sumergir todas las fibras de fibroína usando una espátula. Cubrir el vaso para evitar la evaporación y colocarlo a 60 ° C durante 4 horas para permitir que las fibras se disuelven. (15 minutos) </li><li> El uso de la jeringa, recoja la solución fibroína del vaso de precipitados y cargarlo en los MWCO 3.500 casetes de diálisis. Realizar la diálisis frente a agua destilada durante 48 horas. Cambie el agua cada dos horas. </li><li> El uso de la jeringa, recoger la solución de fibroínalos cassettes en 50 ml tubos cónicos y se centrifuga dos veces a 9.000 rpm (~ 12.700 xg) a 4 ° C durante 20 min. Verter el sobrenadante en un tubo nuevo después de cada etapa de centrifugación y desechar el sedimento. (40 min) </li><li> Medir la concentración de la fibroína mediante la estimación del peso en seco. Vierta 1 ml de solución de seda en una barca pesar. Secar la muestra en el horno a 60 ° C durante 2 hr. Pesar la fibroína de seda seco y calcular la concentración de la solución de fibroína de seda multiplicando el peso obtenido por 100. La concentración esperada de solución de seda es 6-9% (w / v). </li><li> Ajustar la concentración de seda a 6% (w / v) mediante la dilución en agua destilada. Punto de detención: La fibroína de seda líquida se puede almacenar a 4 ° C durante un máximo de un mes, en un recipiente cerrado. </li></ol></li><li> Preparación de andamios porosos de solución de seda. <ol><li> Tamizar la NaCl granular para separar los gránulos de tamaño de 500-600 micras, que se utilizará en pasos posteriores. Deseche el gránulos de tamaño por debajo de 500 micras y por encima de 600 micras. (15 minutos) </li><li> Verter 30 ml de solución de seda 6% en el molde de politetrafluoroetileno (PTFE) (Figura 1). Esparcir suavemente 60 g de tamizado NaCl sobre la seda. Toque en el recipiente para obtener una capa uniforme de sal. Incubar 48 horas a RT para polimerizar el seda. </li><li> Coloque el molde de PTFE que contiene andamio en el horno a 60 ° C durante 1 hora para finalizar la polimerización y se evapore todo el líquido restante. </li><li> Coloque el contenido del molde de PTFE en un vaso de precipitados que contenía 2 L de agua destilada durante 48 horas para lixiviar la sal. Cambie el agua 2-3 veces al día. Retirar los andamios de esponja de los moldes cuando la sal está completamente lixiviado punto de detención:. Las esponjas se pueden almacenar sumergido en agua a 4 ° C en un recipiente cerrado para evitar que los andamios de la deshidratación. </li><li> Cuando esté listo, cortar los andamios con 5 mm punzón de biopsia de diámetro. Precortadas los andamios para alcanzar unos 2 mm de altura. PuNCH ​​a cabo el centro del andamio con el punzón de biopsia 2 mm de diámetro (Figura 2A). (1 hr) </li><li> Autoclave los andamios sumergidas en agua para esterilizarlos (ciclo de mojado, 121 ° C, 20 min) punto de detención:. Las esponjas se pueden almacenar sumergido en agua a 4 ° C en un recipiente cerrado para evitar que los andamios de la deshidratación. </li><li> Antes de la siembra de células planeado, sumerja los andamios en solución estéril 0,1 mg / ml de poli-D-lisina (PDL). Incubar durante 1 hora a 37 ° C. </li><li> Lavar los andamios 3 veces con tampón fosfato salino (PBS) para eliminar PDL no unido. (30 minutos) </li></ol></li></ol> 2. Aislamiento de rata neuronas corticales <ol><li> Diseccionar cortezas del día embrionario 18 ratas (E18) Sprague-Dawley como se describe anteriormente por Pacifici y Peruzzi 27 después de obtener animales protocolo aprobado. (2 hr) </li><li> Incubar 10 cortezas en 5 ml de tripsina al 0,25% con 0,3% de DNasa I (de páncreas bovino) durante 20 min a 37 ° C. </li><li> Inactivar la tripsina mediante la adición de un volumen igual de / proteína de soja ml 1 mg. </li><li> Se tritura las cortezas utilizando 10 ml de pipeta Pasteur por pipeteando arriba y abajo 20 veces hasta que se genera una suspensión de células individuales. Sea amable y evitar la formación de burbujas de aire. (10 minutos) </li><li> Centrifugar la suspensión celular a 127 xg durante 5 min. </li><li> Vuelva a suspender el sedimento celular en 10 ml de medio de cultivo (medio Neurobasal, suplemento de B27 1x, 1x Glutamax, 1% penicilina / estreptomicina). Contar las células. La concentración celular esperada es de aproximadamente 2x10 7 / ml. (20 min) </li></ol> 3. Construir Asamblea y Cultura <ol><li> Andamios siembra con las células. <ol><li> Mueva los andamios estériles y todos los utensilios necesarios dentro de una campana de cultivo celular. Usando pinzas estériles colocan los andamios en una placa de cultivo celular de 96 pocillos de asignar un andamio por pocillo. (10 minutos) </li><li> Sumergir los andamios en medio de cultivo celular para equilibrar ellos antes de la cavidad de la semillaEn g. Incubar durante 1 hora a 37 ° C. (10 minutos) </li><li> Aspirar el exceso de medio a partir de los andamios. </li><li> Aplicar 100 l de suspensión de células / andamio. (10 minutos) </li><li> Se incuban las células a 37 ° CO / N para permitir la unión de las células al andamio. </li><li> A la mañana siguiente aspirar las células no inscritos y aplicar 200 l / pocillo de medio de cultivo fresco. (10 minutos) </li></ol></li><li> Andamio incrustación con matriz de colágeno. (2 hr) <ol><li> Coloque 10x PBS, agua, NaOH 1 N y cola de rata I colágeno en hielo. Preparar una solución de trabajo de colágeno de acuerdo con las instrucciones del fabricante. Mantener en hielo hasta las construcciones de células sembradas están listas (hasta 1 hora). </li><li> Retire las construcciones de seda sembrado de células de la incubadora y aspirar el exceso de medio. </li><li> Usando pinzas estériles transfieren los andamios a los pozos vacíos en la placa de bien y se sumergen cada andamio en 100 l de solución de colágeno 3 mg / ml. Coloque la placa de cultivo de tejidos de regresoen la incubadora durante 30 minutos para permitir la polimerización del colágeno. </li><li> Aplicar 100 l de medio de cultivo celular pre-calentado / pocillo. Cultura los constructos durante una semana cambiando el medio cada día mediante la sustitución de sólo la mitad del volumen del medio. </li></ol></li></ol> 4. Análisis de Microscopía <ol><li> Tinción vivo / muerto (1 hora) <ol><li> Preparar la solución madre de yoduro de propidio (PI) 1 mg / ml (1,5 mM) en agua destilada. </li><li> Preparar solución madre de diacetato de fluoresceína (FDA) 2 mg / ml en acetona. </li><li> Preparar la solución de trabajo que contiene 10 PI M y 0,15 M FDA diluido en PBS. Pre-calentar la solución a 37 ° C en un baño de agua. </li><li> Se lavan las células con PBS precalentado para eliminar las esterasas de suero. Aspirar el PBS. </li><li> Aplique la solución pre-calentado a trabajar en las células durante 2 minutos y lavar con PBS. </li><li> Utilice microscopio de epifluorescencia a la imagen de las células (PI ex λ = 490 nm, em λ = 570 nm; FDA ex λ = 490 nm, em λ = 514 nm). La mancha se mantiene estable durante 40 min. Tinción prolongada puede resultar en la tinción inespecífica que resulta de la captación de PI por las células y co-localización de PI / FDA en las células. </li></ol></li><li> Inmunoticción <ol><li> En el punto de tiempo deseado de la cultura fijar las células con paraformaldehído al 4% (PFA) durante 30 min a TA. Debido a la toxicidad de la PFA humos de la etapa de fijación se debe realizar en la campana de humos química. </li><li> Lave los andamios con 3x PBS punto de detención:. Las construcciones fijas-PFA se pueden almacenar en PBS a 4 ° C durante varios días antes de proceder con nuevas medidas. </li><li> Incubar los andamios en el 0,2% Tritón, 0,25% de albúmina de suero bovino (BSA) en PBS que contenía anticuerpo primario O / N a 4 ° C. </li><li> El día siguiente descarta la solución de tinción y lavar los andamios 3 x 30 min con PBS para eliminar el anticuerpo primario no unido. </li><li> Se incuban las muestras con el anticuerpo secundario diluido en 0,2% Triton, 0,25% de BSA en PBS durante 2hr a RT. </li><li> Retirar la solución de tinción y lavar los andamios 3 x 30 min con PBS para eliminar el anticuerpo secundario no unido. </li><li> Imagen de los andamios utilizando un microscopio confocal con objetivo 20X tomando 100 micras z secciones de la muestra con 1 m paso. Para visualizar las neuritas delgadas la resolución de la imagen debe ser de un mínimo de 1.024 x 1.024 píxeles. </li></ol></li><li> ARN, ADN y el aislamiento de proteínas Nota: RNA, DNA y el aislamiento de proteínas se realiza usando un kit comercial DNA Mini AllPrep / ARN / proteína de acuerdo con las instrucciones del fabricante. <ol><li> Usando pinzas estériles transfieren los andamios de placa de cultivo a un tubo estéril de 2 ml. Coloque un andamio por tubo. </li><li> Añadir 200 l de tampón RLT a cada tubo. </li><li> Interrumpir el constructo fragmentando con micro tijeras estériles. </li><li> Para homogeneizar el tejido interrumpido, transferir el contenido del tubo para la columna de centrifugación. Haga girar durante 2 minutos a toda velocidad en una microcentrífuga.El lisado se homogeneizó a medida que pasa a través de la columna de centrifugación. </li><li> Recoger el flujo a través y utilizarla inmediatamente para aislar RNA, DNA y proteínas siguiendo el protocolo del fabricante. </li><li> Cuantificar el rendimiento de los ácidos nucleicos cualquier otro método compatible (por ejemplo, NanoDrop). </li><li> Cuantificar el rendimiento de proteína utilizando ensayo de proteína BCA de acuerdo con las instrucciones del fabricante. Medir el resultado de ensayo usando lector de microplacas a 562 nm de longitud de onda. Nota: El rendimiento esperado de los ácidos nucleicos y proteína por construcción en relación con la densidad celular se muestra en la Figura 4. </li></ol></li></ol>

<ol>
	<li>Dua, T., Janca, A., Kale, R., Montero, F., Muscetta, A., Peden, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+1,+Public+health+principles+and+neurological+disorders.">Chapter 1, Public health principles and neurological disorders.</a> Neurological Disorders. , (2005).</li><li>Ohtsuki, S., Hirayama, M., Ito, S., Uchida, Y., Tachikawa, M., Teresaki, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=T.+Quantitative+targeted+proteomics+for+understanding+the+blood-brain+barrier:+towards+pharmacoproteomics.">T. Quantitative targeted proteomics for understanding the blood-brain barrier: towards pharmacoproteomics.</a> Expert. Rev. Proteomic. 11 (3), 303-313 (2014).</li><li>Hyder, F., Rothman, D. L., Bennett, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cortical+energy+demands+of+signaling+and+nonsignaling+components+in+brain+are+conserved+across+mammalian+species+and+activity+levels.">Cortical energy demands of signaling and nonsignaling components in brain are conserved across mammalian species and activity levels.</a> Proc Natl Acad Sci USA. 110 (9), 3549-3554 (2013).</li><li>Wesseling, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+combined+metabonomic+and+proteomic+approach+identifies+frontal+cortex+changes+in+a+chronic+phencyclidine+rat+model+in+relation+to+human+schizophrenia+brain+pathology.">A combined metabonomic and proteomic approach identifies frontal cortex changes in a chronic phencyclidine rat model in relation to human schizophrenia brain pathology.</a> Neuropsychopharmacology. 38 (12), 2532-2544 (2013).</li><li>Fawcett, J. W., Housden, E., Smith-Thomas, L., Meyer, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+growth+of+axons+in+three-dimensional+astrocyte+cultures.">The growth of axons in three-dimensional astrocyte cultures.</a> Dev Biol. 135 (2), 449-458 (1989).</li><li>Cullen, D. K., Simon, C. M., LaPlaca, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain+rate-dependent+induction+of+reactive+astrogliosis+and+cell+death+in+three-dimensional+neuronal-astrocytic+co-cultures.">Strain rate-dependent induction of reactive astrogliosis and cell death in three-dimensional neuronal-astrocytic co-cultures.</a> Brain Res. 1158, 103-115 (2007).</li><li>East, E., de Oliveira, D. B., Golding, J. P., Phillips, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alignment+of+astrocytes+increases+neuronal+growth+in+three-dimensional+collagen+gels+and+is+maintained+following+plastic+compression+to+form+a+spinal+cord+repair+conduit.">Alignment of astrocytes increases neuronal growth in three-dimensional collagen gels and is maintained following plastic compression to form a spinal cord repair conduit.</a> Tissue Eng. Part A. 16 (10), 3173-3184 (2010).</li><li>Dubois-Dauphin, M. L., Toni, N., Julien, S. D., Charvet, I., Sundstrom, L. E., Stoppini, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+long-term+survival+of+in+vitro+engineered+nervous+tissue+derived+from+the+specific+neural+differentiation+of+mouse+embryonic+stem+cells.">The long-term survival of in vitro engineered nervous tissue derived from the specific neural differentiation of mouse embryonic stem cells.</a> Biomaterials. 31 (27), 7032-7042 (2010).</li><li>Lancaster, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cerebral+organoids+model+human+brain+development+and+microcephaly.">Cerebral organoids model human brain development and microcephaly.</a> Nature. 501 (7467), 373-379 (2013).</li><li>Hogberg, H. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+a+3D+model+of+human+brain+development+for+studying+gene/environment+interactions.">Toward a 3D model of human brain development for studying gene/environment interactions.</a> Stem Cell Res Ther. 4, S4 (2013).</li><li>Kato-Negishi, M., Morimoto, Y., Onoe, H., Takeuchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Millimeter-sized+neural+building+blocks+for+3D+heterogeneous+neural+network+assembly.">Millimeter-sized neural building blocks for 3D heterogeneous neural network assembly.</a> Adv Healthc Mater. 2 (12), 1564-1570 (2013).</li><li>Watanabe, K., Nakamura, M., Okano, H., Toyama, Y. <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+three-dimensional+culture+of+neural+stem/progenitor+cells+in+collagen+type-1+gel.">Establishment of three-dimensional culture of neural stem/progenitor cells in collagen type-1 gel.</a> Restor Neurol Neurosci. 25 (2), 109-117 (2007).</li><li>Aurand, E. R., Wagner, J. L., Shandas, R., Bjugstad, K. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+formulation+determines+cell+fate+of+fetal+and+adult+neural+progenitor+cells.">Hydrogel formulation determines cell fate of fetal and adult neural progenitor cells.</a> Stem Cell Res. 12 (1), 11-23 (2014).</li><li>Gurkan, U. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=et+al..Simple+precision+creation+of+digitally+specified,+spatially+heterogeneous,+engineered+tissue+architectures.">et al..Simple precision creation of digitally specified, spatially heterogeneous, engineered tissue architectures.</a> Adv Mater. 25 (8), 1192-1198 (2013).</li><li>Mironov, V., Boland, T., Trusk, T., Forgacs, G., Markwald, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+printing:+computer-aided+jet-based+3D+tissue+engineering.">Organ printing: computer-aided jet-based 3D tissue engineering.</a> Trends Biotechnol. 21 (4), 157-161 (2003).</li><li>Taylor, A. M., Blurton-Jones, M., Rhee, S. W., Cribbs, D. H., Cotman, C. W., Jeon, N. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+microfluidic+culture+platform+for+CNS+axonal+injury,+regeneration+and+transport.">A microfluidic culture platform for CNS axonal injury, regeneration and transport.</a> Nature. 2 (8), 599-605 (2005).</li><li>Tang-Schomer, M. D., Davies, P., Graziano, D., Thurber, A. E., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+circuits+with+long-distance+axon+tracts+for+determining+functional+connectivity.">Neural circuits with long-distance axon tracts for determining functional connectivity.</a> J Neurosci Methods. 222, 82-90 (2014).</li><li>Tang-Schomer, M. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioengineered+functional+brain-like+cortical+tissue.">Bioengineered functional brain-like cortical tissue.</a> Proc Natl Acad Sci USA. 111 (38), 13811-13816 (2014).</li><li>Rockwood, D. N., Preda, R. C., Y&#252;cel, T., Wang, X., Lovett, M. L., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Materials+fabrication+from+Bombyx+mori+silk+fibroin.">Materials fabrication from Bombyx mori silk fibroin.</a> Nat Protoc. 6, 1612-1631 (2011).</li><li>Lawrence, B. D., Pan, Z., Weber, M. D., Kaplan, D. L., Rosenblatt, M. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+film+culture+system+for+in+vitro+analysis+and+biomaterial+design.">Silk film culture system for in vitro analysis and biomaterial design.</a> J. Vis. Exp. (62), e3646 (2012).</li><li>Maddah, M., Grimson, W. E., Warfield, S. K., Wells, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+unified+framework+for+clustering+and+quantitative+analysis+of+white+matter+fiber+tracts.">A unified framework for clustering and quantitative analysis of white matter fiber tracts.</a> Med Image Anal. 12 (2), 191-202 (2008).</li><li>Vepari, C., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+as+a+biomaterial.">Silk as a biomaterial.</a> Prog Polym Sci. 32 (8-9), 8-9 (2007).</li><li>Yao, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salt-leached+silk+scaffolds+with+tunable+mechanical+properties.">Salt-leached silk scaffolds with tunable mechanical properties.</a> Biomacromolecules. 13 (11), 3723-3729 (2012).</li><li>Kim, U. J., Park, J., Kim, H. J., Wada, M., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+aqueous-derived+biomaterial+scaffolds+from+silk+fibroin.">Three-dimensional aqueous-derived biomaterial scaffolds from silk fibroin.</a> Biomaterials. 26 (15), 2775-2785 (2005).</li><li>Gil, E. S., Mandal, B. B., Park, S. H., Marchant, J. K., Omentetto, F. G., Kaplan, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Helicoidal+multi-lamellar+features+of+RGD-functionalized+silk+biomaterials+for+corneal+tissue+engineering.">Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering.</a> Biomaterials. 31 (34), 8953-8963 (2010).</li><li>Hopkins, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silk+hydrogels+as+soft+substrates+for+neural+tissue+engineering.">Silk hydrogels as soft substrates for neural tissue engineering.</a> Adv Funct Mater. 23 (41), 5140-5149 (2013).</li><li>Pacifici, M., Peruzzi, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+culture+of+rat+embryonic+neural+cells:+A+quick+protocol.">Isolation and culture of rat embryonic neural cells: A quick protocol.</a> Vis. Exp. (63), e3965 (2012).</li></ol>

La publicación de este video-artículo es patrocinado por Corning.

Here we described the guidelines to assemble a compartmentalized 3D brain-like tissue model. The model is characterized by dense polarized culture of neurons resulting in the development of two morphologically different compartments: one containing densely packed cell bodies, second containing pure axonal networks. Overall, the scaffold architecture and growth pattern mimic brain cortical tissue including the six laminar layers and white matter tracts21.
The donut-shaped silk protein-based tissue model allows for modifications and tuning of mechanical properties, versatile structural forms, hydrogel fillers and cellular components. Thus, this tissue model establishes a base platform for a wide range of studies. Silk is a favorable candidate for biomaterial platforms due to its biocompatibility, aqueous-based processing, and robust mechanical and degradation properties22. The silk matrix also serves as a stable anchor to reduce collagen gel contraction over time in culture. The properties such as silk concentration and porosity of the scaffold used in this model have been previously adjusted to achieve optimal cell growth and mechanical properties resulting in brain-like tissue elasticity18,23,24. We suggest to keep these parameters constant to ensure the successful outcome and reproducibility of the experiments.
The 3D neuronal network formation was achieved by combining two types of biomaterials with different mechanical properties: a stiff scaffold to provide neuronal anchoring and a softer gel matrix to permit axon penetration and connectivity in 3D. Selective material preferences of the silk scaffold and soft hydrogel provide the underlying principle for compartmentalizing the neurons from the axonal connections. Due to the inert nature of the silk scaffold, functionalization with poly-D-lysine is required for neuronal attachment. However, other cell adhesion promoting factors can be applied such as RGD25 or fibronectin26. To achieve the 3D network formation the silk scaffold needs to be filled with hydrogel in a timely manner as soon as neurons are attached to the scaffold. Likewise, the collagen matrix filler can be replaced by other hydrogels, thus allowing the study of the influence of 3D extracellular environments on axonal network formation to serve as a platform to evaluate novel hydrogels in terms of neuronal compatibility. Additionally, apart from rat cortical neurons other neuronal sources such as hippocampal neurons or induced pluripotent cell (iPSc)-derived neurons can be utilized. Moreover, the tissue model can be used to study heterocellular interactions by including glial cells along with neurons and to build more complex brain-like tissue.
As shown previously, our model can be utilized to evaluate neuronal functionality in 3D microenvironment with a variety of assays such as cell viability, gene expression, LC-MS/MS and electrophysiology, thus demonstrating physiological relevance of the model18. Other methods, which are frequently utilized to evaluate 3D tissue-engineered constructs, such as immunostaining and microscopy8,9,11 can also be used to assess cell distribution and extent of axonal network formation. However, it has to be noted, that due to the high density of the collagen matrix, the penetration of antibodies and depth of the imaging is limited to few hundred micrometers. Moreover, the signal to noise ratio may be affected by nonspecific background fluorescence. This can be overcome by using lipophilic cell tracers and genetically expressed fluorescent proteins which diminish the need for immunostaining11. Alternatively, the imaging can be performed with 2-photon microscopy instead of usual one-photon technique, which may reduce the signal loss, photobleaching, and can be extended to several hundred micrometers of depth.
Summarizing, the silk and collagen-based brain-like tissue offers a robust platform to study neuronal networks in 3D and is a starting point for the development of more advanced models of neurological disorders in the future. Independent from the mode of evaluation, the relative simplicity of this tissue model supports its utility, success and reproducibility.

El sistema nervioso central (CNS) puede ser afectada por una variedad de trastornos que implican vascular, estructural, funcional, infeccioso o degenerativo. Se estima que unos 6,8 millones de personas mueren cada año como consecuencia de los trastornos neurológicos, lo que representa una carga socioeconómica en crecimiento en todo el mundo 1. Sin embargo, sólo unos pocos de los trastornos tienen tratamientos disponibles. Por lo tanto, hay una necesidad crítica de estrategias terapéuticas innovadoras para pacientes que sufren de trastornos neurológicos. Desafortunadamente, muchas terapias orientados SNC fracasan durante los ensayos clínicos, en parte debido a la utilización de los modelos de investigación pre-clínicos inadecuados, que no permiten la evaluación de los efectos agudos y crónicos con lecturas funcionales fisiológicamente relevantes. A pesar de los esfuerzos de investigación significativos en las últimas décadas, hay una gran cantidad desconocida acerca de la estructura y función del sistema nervioso central. Con el fin de obtener más conocimientos, modelos animales son frecuentemente nosed modelar estados patológicos, tales como lesión cerebral traumática (TBI) o demencia, sobre todo en los estudios preclínicos. Sin embargo, los animales difieren significativamente de los seres humanos, tanto en la anatomía del sistema nervioso central, así como en función, expresión génica y el metabolismo 2-4. Por otra parte, los cultivos 2D in vitro son el método común para investigar la biología celular y se utilizan de forma rutinaria para el descubrimiento de fármacos. Sin embargo, los cultivos celulares 2D carecen de la complejidad y la relevancia fisiológica en comparación con cerebro humano 5-7. Mientras que no hay sustituto para el bajo costo y la sencillez de los estudios de cultivo celular en 2D o la complejidad proporcionada por los modelos animales, la ingeniería de tejidos 3D podría generar mejores modelos de investigación para cerrar la brecha que existe entre el 2D in vitro e in vivo enfoques. La ingeniería de tejidos 3D proporciona condiciones experimentales más fisiológicamente relevantes obtenidos por las interacciones célula-célula en 3D y señales extracelulares proporcionadas por los biomateriales. Despite la evidencia significativa detrás del valor de las culturas en 3D, en la actualidad hay sólo unos pocos modelos de tejido 3D SNC como madre derivadas de células culturas organoides 8-10, neurospheroids 11 y se dispersaron culturas hidrogel 12,13. Métodos técnicos avanzados, incluyendo la litografía de múltiples capas 14, y la impresión 3D 15 se han utilizado para el estudio de los pulmones, el hígado y el tejido renal. Sin embargo, hay una falta de modelos 3D del SNC que permitan el crecimiento neuronal compartimentada, como imitando la arquitectura cortical y la biología. Separado el crecimiento de las neuritas de los cuerpos celulares neuronales se ha demostrado previamente en cultivos 2D mediante el uso de microfabricación 16,17 permitiendo el estudio de rastreo tracto axón, el influjo de calcio, arquitectura de red y actividades. Esta idea nos inspiró para desarrollar un tejido neural polarizado 3D donde se encuentran los cuerpos celulares y tractos axonales en diferentes compartimentos que imitan la arquitectura de capas del cerebro 18 </sup&gt;. Nuestro enfoque se basa en el uso de diseño único andamio de seda que tiene capacidad de alta densidad de células en un volumen confinado y permite consecuencia de densa red axonal en un gel de colágeno. Aquí se demuestra el procedimiento de montaje completo del tejido cerebral como incluyendo la fabricación de andamios y la cultura neuronal.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Na2CO3</td>
			<td>Sigma-Aldrich</td>
			<td>223530</td>
			<td>-</td>
		</tr>
		<tr>
			<td>LiBr</td>
			<td>Sigma-Aldrich</td>
			<td>213225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>MWCO 3500 dialysis cassettes&#160;</td>
			<td>Thermo Fisher</td>
			<td>66110</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Heating plate</td>
			<td>Fisher Scientific</td>
			<td>Isotemp</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Centrifuge 5804 R</td>
			<td>Eppendorf</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Sieve</td>
			<td>Fisher Scientific</td>
			<td>-</td>
			<td>No. 270, No. 35, No. 30</td>
		</tr>
		<tr>
			<td>PTFE mold</td>
			<td>-</td>
			<td>-</td>
			<td>made in house (Figure 1) 10 cm diameter, 2 cm height</td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Sigma-Aldrich</td>
			<td>71382</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 5mm</td>
			<td>World Precision Instrument</td>
			<td>501909</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Biopsy Punch 2mm</td>
			<td>World Precision Instrument</td>
			<td>501908</td>
			<td>-</td>
		</tr>
		<tr>
			<td>poly-D-lysine</td>
			<td>Sigma-Aldrich</td>
			<td>P6407-5MG</td>
			<td>final concentration 10 ug/ml, dissolved in water</td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Sigma-Aldrich</td>
			<td>D1283-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma-Aldrich</td>
			<td>T4049-500ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>DNase</td>
			<td>Roche</td>
			<td>10104159001</td>
			<td>final concentration 0.3%</td>
		</tr>
		<tr>
			<td>Soybean protein</td>
			<td>Sigma-Aldrich</td>
			<td>T6522-100MG</td>
			<td>final concentration 1 mg/ml</td>
		</tr>
		<tr>
			<td>Neurobasal medium</td>
			<td>Gibco</td>
			<td>21103049</td>
			<td>warm up to 37&#176;C before use</td>
		</tr>
		<tr>
			<td>B27 supplement 50x</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Glutamax</td>
			<td>Gibco</td>
			<td>35050-061</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Penicilin Streptomycin</td>
			<td>Corning</td>
			<td>30-002-CI</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Collagen I, rat tail, 100 mg</td>
			<td>Corning</td>
			<td>354236</td>
			<td>final concentration 3 mg/ml</td>
		</tr>
		<tr>
			<td>NaOH</td>
			<td>Sigma-Aldrich</td>
			<td>S2770</td>
			<td>corrosive</td>
		</tr>
		<tr>
			<td>Propidium Iodide</td>
			<td>Sigma-Aldrich</td>
			<td>P4170-10MG</td>
			<td>toxic</td>
		</tr>
		<tr>
			<td>Fluorescein Diacetate</td>
			<td>Sigma-Aldrich</td>
			<td>F7378-5G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Olympus MVX10</td>
			<td>Olympus</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Paraformaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>P6148</td>
			<td>toxic, final concentration 4%</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906-10G</td>
			<td>-</td>
		</tr>
		<tr>
			<td>anti-&#946;IIITubulin antibody&#160;</td>
			<td>Sigma-Aldrich</td>
			<td>T2200</td>
			<td>rabbit 1:500</td>
		</tr>
		<tr>
			<td>anti-rabbit Alexa-546</td>
			<td>Molecular Probes</td>
			<td>A11010</td>
			<td>goat 1:250</td>
		</tr>
		<tr>
			<td>goat serum</td>
			<td>Sigma-Aldrich</td>
			<td>G9023-5ML</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Leica SP2 confocal microscope</td>
			<td>Leica</td>
			<td>-</td>
			<td>objective 20x</td>
		</tr>
		<tr>
			<td>QIAshredder</td>
			<td>Qiagen</td>
			<td>79654</td>
			<td>-</td>
		</tr>
		<tr>
			<td>AllPrep DNA/RNA/Protein Mini Kit</td>
			<td>Qiagen</td>
			<td>80004</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, Pure</td>
			<td>Sigma-Aldrich</td>
			<td>E7023&#160;</td>
			<td>-</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>Sigma-Aldrich</td>
			<td>63689</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000c/2000 UV-Vis Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>-</td>
			<td>-</td>
		</tr>
		<tr>
			<td>BCA protein assay</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Plate reader Spectramax M2</td>
			<td>Molecular Devices</td>
			<td>-</td>
			<td>absorbance 562 nm</td>
		</tr>
		<tr>
			<td>Micro Scissors, Economy, Vannas-type</td>
			<td>TedPella</td>
			<td>1346</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

engineered 3d silk-collagen-based model of polarized neural tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Solid esponjas de seda de precorte a la forma de rosquilla representa una idea única y simple para lograr una arquitectura compartimentada se asemeja tejido neural (Figura 2A). La estructura altamente porosa del andamio con el tamaño de poro de aproximadamente 500 micras en el área de resultados excepcionalmente alta superficie que permiten la siembra y el crecimiento de células de alta densidad dentro de un pequeño volumen (2x10 7 / ml) (Figura 2B). Además, la alta porosidad del andamio permite la difusión irrestricta de nutrientes y desechos resultantes de la viabilidad superior de los cultivos de células densas más de marcos de tiempo extendidos. Tras la siembra de las neuronas adjuntar rápida y uniformemente a la superficie de los poros de andamios. Después de la unión celular es completa y las células se equilibran en el sustrato de seda, el andamio esponja se llena con la matriz de colágeno suave con el fin de proporcionar un entorno 3D para la formación de la red axonal (Figura 3 </strong&gt;). En ausencia de la matriz de colágeno de la compartimentación de los axones y cuerpos celulares no es posible, ya que las neuronas crecen extensiones sólo en la superficie de los poros que resultan en redes mixtas como se encuentra con las superficies 2D (Figura 3B). En contraste, el gel de colágeno proporciona una malla que soporta extensa crecimiento axonal en 3D, mientras que los cuerpos celulares neuronales permanecer unidos a la andamio de seda (Figura 3C). Este patrón de crecimiento conduce a la compartimentación de los cuerpos celulares y redes axonal puros (Figura 3D), que se asemeja a la materia gris y blanca de la corteza del cerebro. Aparte de microscopía, las construcciones se pueden evaluar con una variedad de otros métodos 18, dependiendo de la pregunta detrás del experimento. Por ejemplo, el aislamiento de ADN, ARN y proteínas de las construcciones se puede realizar fácilmente utilizando kits comerciales (Figura 4). El rendimiento de ácidos nucleicos y de proteínas PEr constructo depende del número de células sembradas inicialmente en los andamios de seda. Los más células sembradas mayor es el rendimiento de ADN, ARN y proteínas. <img alt="Figura 1" src="/files/ftp_upload/52970/52970fig1.jpg" /> Figura 1. molde de PTFE utilizado para la preparación de andamios esponja de seda Las dimensiones:.. 10 cm de diámetro, 2 cm de altura <a href="https://www.jove.com/files/ftp_upload/52970/52970fig1large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 2" src="/files/ftp_upload/52970/52970fig2.jpg" /> Figura 2. 3D modelo de tejido cerebral similares. (A) poroso andamio esponja seda. (B) en vivo tinción / muertos de las neuronas en el día 1 en la siembra en andamio de seda antes de colágeno incrustar (células-verde vivo, células de color rojo-muertos). Los paneles en el lado derecho muestran la magnification del área capturada en cuadros rojos. <a href="https://www.jove.com/files/ftp_upload/52970/52970fig2large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura.</a> <img alt="Figura 3" src="/files/ftp_upload/52970/52970fig3.jpg" /> Figura 3. Neuronal consecuencia compartimentada en el modelo de tejido cerebral como 3D (A) compartimentos de andamios:. Compartimiento del cuerpo de las células rojas, compartimento azul-axonal. (B) el patrón de crecimiento neuronal en el día 1 tras la siembra antes de la incrustación de colágeno. (C). Consecuencia neuronal en el día 7 tras la siembra en el compartimiento del cuerpo celular. (D) consecuencia neuronal en el día 7 tras la siembra en el compartimiento axonal. Verde -. ΒIIITubulin <a href="https://www.jove.com/files/ftp_upload/52970/52970fig3large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta</a>cifra. <img alt="Figura 4" src="/files/ftp_upload/52970/52970fig4.jpg" /> Figura 4. Rendimiento esperado de (A) ARN y ADN y proteínas (B) por construcción en relación con la cantidad de células sembradas por andamio (± DE, n = 5). <a href="https://www.jove.com/files/ftp_upload/52970/52970fig4large.jpg" target="_blank">Haga clic aquí para ver una versión más grande de esta figura .</a>

Watch this Scientific Journal Video about Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue at JoVE.com

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

engineered-3d-silk-collagen-based-model-of-polarized-neural-tissue

Research

JoVE Journal

Bioengineering

Electric Field-controlled Directed Migration of Neural Progenitor Cells in 2D and 3D Environments

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the central nervous system1,2. These endogenous EFs are generated by cellular regulation of ionic transport combined with the electrical resistance of cells and tissues. It has been reported that applied EF treatment can promote functional repair of spinal cord injuries in animals and humans3,4. In particular, EF-directed cell migration has been demonstrated in a wide variety of cell types5,6, including neural progenitor cells (NPCs)7,8. Application of direct current (DC) EFs is not a commonly available technique in most laboratories. We have described detailed protocols for the application of DC EFs to cell and tissue cultures previously5,11. Here we present a video demonstration of standard methods based on a calculated field strength to set up 2D and 3D environments for NPCs, and to investigate cellular responses to EF stimulation in both single cell growth conditions in 2D, and the organotypic spinal cord slice in 3D. The spinal cordslice is an ideal recipient tissue for studying NPC ex vivo behaviours, post-transplantation, because the cytoarchitectonic tissue organization is well preserved within these cultures9,10. Additionally, this ex vivo model also allows procedures that are not technically feasible to track cells in vivo using time-lapse recording at the single cell level. It is critically essential to evaluate cell behaviours in not only a 2D environment, but also in a 3D organotypic condition which mimicks the in vivo environment. This system will allow high-resolution imaging using cover glass-based dishes in tissue or organ culture with 3D tracking of single cell migration in vitro and ex vivo and can be an intermediate step before moving onto in vivo paradigms.

This protocol demonstrates methods used to establish 2D and 3D environments in custom-designed electrotactic chambers, which can track cells in vivo/ex vivo using time-lapse recording at the single cell level, in order to investigate galvanotaxis/electrotaxis and other cellular responses to direct current (DC) electric fields (EFs).

Campo eléctrico controlado por migración dirigida de células progenitoras neurales en entornos 2D y 3D

Endogenous electric fields (EFs) occur naturally in vivo and play a critical role during tissue/organ development and regeneration, including that of the ...

Investigación

Bioingeniería

Magnetic Resonance Elastography Methodology for the Evaluation of Tissue Engineered Construct Growth

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use of destructive assessment is not acceptable. A proposed alternative is the use of an imaging process called magnetic resonance elastography. Elastography is a nondestructive method for determining the engineered outcome by measuring local mechanical property values (i.e., complex shear modulus), which are essential markers for identifying the structure and functionality of a tissue. As a noninvasive means for evaluation, the monitoring of engineered constructs with imaging modalities such as magnetic resonance imaging (MRI) has seen increasing interest in the past decade1. For example, the magnetic resonance (MR) techniques of diffusion and relaxometry have been able to characterize the changes in chemical and physical properties during engineered tissue development2. The method proposed in the following protocol uses microscopic magnetic resonance elastography (&mu;MRE) as a noninvasive MR based technique for measuring the mechanical properties of small soft tissues3. MRE is achieved by coupling a sonic mechanical actuator with the tissue of interest and recording the shear wave propagation with an MR scanner4. Recently, &mu;MRE has been applied in tissue engineering to acquire essential growth information that is traditionally measured using destructive mechanical macroscopic techniques5. In the following procedure, elastography is achieved through the imaging of engineered constructs with a modified Hahn spin-echo sequence coupled with a mechanical actuator. As shown in Figure 1, the modified sequence synchronizes image acquisition with the transmission of external shear waves; subsequently, the motion is sensitized through the use of oscillating bipolar pairs. Following collection of images with positive and negative motion sensitization, complex division of the data produce a shear wave image. Then, the image is assessed using an inversion algorithm to generate a shear stiffness map6. The resulting measurements at each voxel have been shown to strongly correlate (R2&gt;0.9914) with data collected using dynamic mechanical analysis7. In this study, elastography is integrated into the tissue development process for monitoring human mesenchymal stem cell (hMSC) differentiation into adipogenic and osteogenic constructs as shown in Figure 2.

The procedure demonstrates the methodology of magnetic resonance elastography for monitoring the engineered outcome of adipose and osteogenic tissue engineered constructs through noninvasive local assessment of the mechanical properties using microscopic magnetic resonance elastography (&mu;MRE).

Metodología elastografía por resonancia magnética para la Evaluación de los tejidos Diseñado crecimiento Construct

Traditional mechanical testing often results in the destruction of the sample, and in the case of long term tissue engineered construct studies, the use ...

Protocol for Relative Hydrodynamic Assessment of Tri-leaflet Polymer Valves

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of tri-leaflet polymer valve prostheses. However, identification of a protocol for initial assessment of polymer valve hydrodynamic functionality is paramount during the early stages of the design process. Traditional in vitro pulse duplicator systems are not configured to accommodate flexible tri-leaflet materials; in addition, assessment of polymer valve functionality needs to be made in a relative context to native and prosthetic heart valves under identical test conditions so that variability in measurements from different instruments can be avoided. Accordingly, we conducted hydrodynamic assessment of i) native (n = 4, mean diameter, D = 20 mm), ii) bi-leaflet mechanical (n= 2, D = 23 mm) and iii) polymer valves (n = 5, D = 22 mm) via the use of a commercially available pulse duplicator system (ViVitro Labs Inc, Victoria, BC) that was modified to accommodate tri-leaflet valve geometries. Tri-leaflet silicone valves developed at the University of Florida comprised the polymer valve group. A mixture in the ratio of 35:65 glycerin to water was used to mimic blood physical properties. Instantaneous flow rate was measured at the interface of the left ventricle and aortic units while pressure was recorded at the ventricular and aortic positions. Bi-leaflet and native valve data from the literature was used to validate flow and pressure readings. The following hydrodynamic metrics were reported: forward flow pressure drop, aortic root mean square forward flow rate, aortic closing, leakage&#160;and regurgitant volume, transaortic closing, leakage, and total energy losses. Representative results indicated that hydrodynamic metrics from the three valve groups could be successfully obtained by incorporating a custom-built assembly into a commercially available pulse duplicator system and subsequently, objectively compared to provide insights on functional aspects of polymer valve design.

There has been renewed interest in developing polymer valves. Here, the objectives are to demonstrate the feasibility of modifying a commercial pulse duplicator to accommodate tri-leaflet geometries and to define a protocol to present polymer valve hydrodynamic data in comparison to native and prosthetic valve data collected under near-identical conditions.

Protocolo para la evaluación relativa hidrodinámico de Tri-lámina polimérica Válvulas

Limitations of currently available prosthetic valves, xenografts, and homografts have prompted a recent resurgence of developments in the area of ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Ingeniería de tejidos de un humano 3D<em&gt; In vitro</em&gt; Sistema de Prueba de Tumores

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Generation and Grafting of Tissue-engineered Vessels in a Mouse Model

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. The current protocol presents an efficient and reproducible strategy in which functional tissue engineered vessel grafts can be generated using partially induced pluripotent stem cell (PiPSC) from human fibroblasts. We designed a decellularized vessel scaffold bioreactor, which closely mimics the matrix protein structure and blood flow that exists within a native vessel, for seeding of PiPSC-endothelial cells or smooth muscle cells prior to grafting into mice. This approach was demonstrated to be advantageous because immune-deficient mice engrafted with the PiPSC-derived grafts presented with markedly increased survival rate 3 weeks after surgery. This protocol represents a valuable tool for regenerative medicine, tissue engineering and potentially patient-specific cell-therapy in the near future.

Here, we present a protocol to generate tissue engineered vessel grafts that are functional for grafting into mice by double seeding partially induced pluripotent stem cell (PiPSC) - derived smooth muscle cells and PiPSC - derived endothelial cells on a decellularized vessel scaffold bioreactor.

Generación y Acrecentamiento de buques de ingeniería de tejidos en un modelo de ratón

The construction of vascular conduits is a fundamental strategy for surgical repair of damaged and injured vessels resulting from cardiovascular diseases. ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

Producción, Caracterización y posibles usos de un Humano esofágica mucosas Modelo ingeniería tisular 3D

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific mutational background. The model is generated on a decellularized porcine scaffold that reproduces tissue-specific characteristics regarding extracellular matrix composition and architecture including the basement membrane. We standardized a protocol that allows artificial tumor tissue generation within 14 days including three days of drug treatment. Our article provides several detailed descriptions of 3D read-out screening techniques like the determination of the proliferation index Ki67 staining&#39;s, apoptosis from supernatants by M30-ELISA and assessment of epithelial to mesenchymal transition (EMT), which are helpful tools for evaluating the effectiveness of therapeutic compounds. We could show compared to 2D culture a reduction of proliferation in our 3D tumor model that is related to the clinical situation. Despite of this lower proliferation, the model predicted EGFR-targeted drug responses correctly according to the biomarker status as shown by comparison of the lung carcinoma cell lines HCC827 (EGFR -mutated, KRAS wild-type) and A549 (EGFR wild-type, KRAS-mutated) treated with the tyrosine-kinase inhibitor (TKI) gefitinib. To investigate drug responses of more advanced tumor cells, we induced EMT by long-term treatment with TGF-beta-1 as assessed by vimentin/pan-cytokeratin immunofluorescence staining. A flow-bioreactor was employed to adjust culture to physiological conditions, which improved tissue generation. Furthermore, we show the integration of drug responses upon gefitinib treatment or TGF-beta-1 stimulation - apoptosis, proliferation index and EMT - into a Boolean in silico model. Additionally, we explain how drug responses of tumor cells with a specific mutational background and counterstrategies against resistance can be predicted. We are confident that our 3D in vitro approach especially with its in silico expansion provides an additional value for preclinical drug testing in more realistic conditions than in 2D cell culture.

A Combined 3D Tissue Engineered In Vitro/In Silico Lung Tumor Model for Predicting Drug Effectiveness in Specific Mutational Backgrounds

We present a three-dimensional (3D) lung cancer model based on a biological collagen scaffold to study sensitivity towards non-small-cell-lung-cancer-(NSCLC)-targeted therapies. We demonstrate different read-out techniques to determine the proliferation index, apoptosis and epithelial-mesenchymal transition (EMT) status. Collected data are integrated into an in silico model for prediction of drug sensitivity.

Un Tejido Combinada 3D Diseñado<em&gt; In Vitro</em&gt; /<em&gt; In silico</em&gt; Modelo de tumor de pulmón para la predicción de la eficacia de drogas en Fondos Específicos mutacional

In the present study, we combined an in vitro 3D lung tumor model with an in silico model to optimize predictions of drug response based on a specific ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Bioprinting de cartílago y piel análogos de tejido utilizando un pasivo novela mezcla de unidad técnica de Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Seeding and Implantation of a Biosynthetic Tissue-engineered Tracheal Graft in a Mouse Model

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional tissue. Tissue engineering holds great promise as a potential solution with its ability to integrate cells and signaling molecules into a 3-dimensional scaffold. Recent work with tissue engineered tracheal grafts (TETGs) has seen some success but their translation has been limited by graft stenosis, graft collapse, and delayed epithelialization. In order to investigate the mechanisms driving these issues, we have developed a mouse model for tissue engineered tracheal graft implantation. TETGs were constructed using electrospun polymers polyethylene terephthalate (PET) and polyurethane (PU) in a mixture of PET and PU (20:80 percent weight). Scaffolds were then seeded using bone marrow mononuclear cells isolated from 6-8 week-old C57BL/6 mice by gradient centrifugation. Ten million cells per graft were seeded onto the lumen of the scaffold and allowed to incubate overnight before implantation between the third and seventh tracheal rings. These grafts were able to recapitulate the findings of stenosis and delayed epithelialization as demonstrated by histological analysis and lack of Keratin 5 and Keratin 14 basal epithelial cells on immunofluorescence. This model will serve as a tool for investigating cellular and molecular mechanisms involved in host remodeling.

Graft stenosis poses a critical obstacle in tissue engineered airway replacement. To investigate cellular mechanisms underlying stenosis, we utilize a murine model of tissue engineered tracheal replacement with seeded bone marrow mononuclear cells (BM-MNC). Here, we detail our protocol, including scaffold manufacturing, BM-MNC isolation, graft seeding, and implantation.

Siembra e implantación de un injerto traqueal tejido-dirigida biosintético en un modelo murino

Treatment options for congenital or secondary long segment tracheal defects have historically been limited due to an inability to replace functional ...

Tissue-Engineered Graft for Circumferential Esophageal Reconstruction in Rats

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant technique with nutritional support. Recently, there have been many attempts at esophageal tissue engineering, but the success rate has been limited due to difficulty in early epithelization in the special environment of peristalsis. Here, we developed an artificial esophagus that can improve the regeneration of the esophageal mucosa and muscle layers through a two-layered tubular scaffold, a mesenchymal stem cell-based bioreactor system, and a bypass feeding technique with modified gastrostomy. The scaffold is made of polyurethane (PU) nanofibers in a cylindrical shape with a three-dimensional (3D) printed polycaprolactone strand wrapped around the outer wall. Prior to transplantation, human-derived mesenchymal stem cells were seeded into the lumen of the scaffold, and bioreactor cultivation was performed to enhance cellular reactivity. We improved the graft survival rate by applying surgical anastomosis and covering the implanted prosthesis with a thyroid gland flap, followed by temporary nonoral gastrostomy feeding. These grafts were able to recapitulate the findings of initial epithelialization and muscle regeneration around the implanted sites, as demonstrated by histological analysis. In addition, increased elastin fibers and neovascularization were observed in the periphery of the graft. Therefore, this model presents a potential new technique for circumferential esophageal reconstruction.

Esophageal reconstruction is a challenging procedure, and development of a tissue-engineered esophagus that enables regeneration of esophageal mucosa and muscle and that can be implanted as an artificial graft is necessary. Here, we present our protocol to generate an artificial esophagus, including scaffold manufacturing, bioreactor cultivation, and various surgical techniques.

Injerto con ingeniería de tejido para la reconstrucción esofágica circunferencial en ratas

The use of biocompatible materials for circumferential esophageal reconstruction is a technically challenging task in rats and requires an optimal implant ...