Los autores desean agradecer a la financiación del Ministerio de Educación ACRF Tier 1 (RG41) y puesta en marcha beca de la Universidad Tecnológica de Nanyang. Bajo y Tjin son financiados por la Beca Estudiantil de Investigación (RSS) de la Universidad Tecnológica de Nanyang, Singapur.

1. Clonación de plásmidos que codifican recombinante para AECM Proteínas <ol><li> Diseño de la secuencia de aminoácidos del dominio funcional (por ejemplo, el dominio de unión celular y elastina repeticiones similares). Sitios de restricción Diseño flanquean los extremos de los dominios funcionales para facilitar la sub-clonación usando software libre de acuerdo con las instrucciones del software (por ejemplo, http://biologylabs.utah.edu/jorgensen/wayned/ape/). Aquí, seleccionar sitios de restricción únicos que no están presentes en el dominio funcional para confinar la digestión a los sitios previstos. Elige sitios de restricción que aparecen en el sitio de clonación múltiple (MCS) del vector de host (por ejemplo, pET22b (+)) para sub-clonación. </li><li> Invertir traducir la secuencia de aminoácidos en la secuencia de nucleótidos utilizando sitios web gratuitos acuerdo con las instrucciones de software. (Por ejemplo, http://www.bioinformatics.org/sms2/rev_trans.html). Asegurarse de codones están optimizados para E. anfitriones coli. </li><li> Gen de interés puede ser purchasoligonucleótidos ed comercialmente como una sola cadena (es decir, si los oligonucleótidos &lt;100 pares de bases (pb)) y realizar el recocido de ADN (véase el paso 1.4) para reducir el costo. De lo contrario, los genes de más de 100 pb, que podían ser adquiridos a través de empresas comerciales. </li><li> Recocido de ADN de los oligonucleótidos <ol><li> Recocer la oligómeros de ADN para obtener la secuencia del gen deseado. Disolver oligonucleótidos de ADN en un tampón de oligómero de ADN (mM Tris 10: 2-amino-2- (hidroximetil) propan-1,3-diol, pH 8,0, filtrada) a una concentración final de 1 g / l. </li><li> Añadir 4 l de cada oligómero a 32 l de tampón de hibridación de ADN (Tris 10 mM, NaCl 100 mM y 100 nM de MgCl 2) para conseguir un total de mezcla de 40 l. </li><li> Hervir un vaso de precipitados de agua utilizando una placa caliente y sumergir la mezcla durante 5 min, 95 ° C. Retire el vaso de precipitados y enfriar gradualmente la totalidad de configurar en una caja de espuma de poliestireno O / N. Los oligómeros han sido recocidas y están listos para la digestión. </li></ol></li&gt; <li> Añadir las enzimas de restricción correspondientes a digerir los oligómeros hibridados (es decir, referido como inserto) y vector huésped (es decir, pET22b (+)) por separado. Utilice la siguiente receta (1-2 g de ADN, 2 l de cada enzima de restricción, 5 l de tampón 10x enzima de restricción, y agua hasta un total mezcla de 50 l) durante 3-4 horas a 37 ° C. NOTA: El pET22b (+) plásmido vector es resistente a los antibióticos ampicilina y contiene un 6x-His tag en el extremo C-terminal. El 6x-His presente en pET22b (+) nos permite utilizar sus-etiquetados anticuerpos para identificar la proteína diana a través de Western Blot en el paso 7. </li><li> Agregar colorante de carga 6x a cada mezcla de digestión. Ejecutar las mezclas de digestión por separado incluyendo una escalera de ADN en 1.2% geles de agarosa de ADN que contiene una mancha de ADN fluorescente UV durante 1 hora a 100 V. visualizar el DNA en gel de agarosa 1,2% con un iluminador de luz UV. </li><li> Cortar el gel para extraer productos de ADN digeridos utilizando kits de purificación de gel comercial. Elute utilizando el volumen mínimo de la columna para lograr una concentración mínima de ADN de 50-100 ng / l. </li><li> Combinar el gen de interés secuencialmente ligando el inserto de ADN digerido (es decir, repeticiones de elastina o dominios de unión de células obtenidos por recocido de ADN) en el vector de plásmido usando la ligasa de T4 utilizando la siguiente receta: (2 l de vector, 1 l de T4 ligasa, 1,5 l de tampón de ligación T4, x l de inserto, 10,5 x-l de agua para un total mezcla de 15 l). Incubar la mezcla de ligación a RT durante 2 hr. NOTA: la concentración molar del vector para insertar debe variar para optimizar la eficiencia de la ligadura. Volumen de inserto descrito como x en la receta depende de la concentración de ADN eluido de la etapa 1.7. </li><li> Descongele E. células químicamente competentes coli DH5a (o cualquier cepa de clonación) en hielo. Placas de agar 2xYT caliente (Tabla 1) que contiene ampicilina (25 mg / ml) a 37 ° C. </li><li> Transformar las células utilizandoGolpe de calor: <ol><li> Alícuota de 50 l de células competentes en tubos de microcentrífuga limpio, pre-refrigerados. Pipetear 5 l (entre 100 pg a 100 ng) de la mezcla de unión en las células, pipetear suavemente hacia arriba y hacia abajo para mezclar. Deje la mezcla en hielo durante 20 min. </li><li> Sumergir el tubo de micro-centrífuga que contiene la mezcla de células en un baño de agua 42 ° C durante 2 min y vuelto a en hielo durante 2 min. Tiempo de la duración de inmersión para minimizar el daño por calor a las células. </li><li> Añadir 500 l de medio SOC (Tabla 1) en el tubo de microcentrífuga y se incuba a 37 ° C con agitación durante 1 hr. </li><li> Spread 50 500 l de la mezcla de células / ligadura sobre una placa de agar 2xYT que ha sido pre-calentado a RT, que contiene ampicilina (25 mg / ml) e incubar las placas al revés a 37 ° CO / N (12-16 hr) . </li></ol></li><li> Al día siguiente, escoja colonias de ADN de la placa de agar utilizando puntas de pipeta limpias. Cultivar las colonias en 5 ml de medio 2YT (Tabla1) que contiene ampicilina (25 mg / ml) O / N a 37 ° CO / N (12-16 hr) con agitación (225 rpm). </li><li> Al día siguiente, utilice un kit de aislamiento de plásmido para extraer los plásmidos de ADN para cada colonia elegido de acuerdo con el protocolo del fabricante. Eluir el ADN con 50 l de agua. </li><li> Realice una prueba de digerir con enzimas de restricción utilizando la siguiente receta: (ADN 5 l, 0,2 l de cada enzima de restricción, de amortiguamiento de la enzima 1 l 10x restricción y recarga de agua para un total mezcla de 10 l), se incuba durante 2 horas a 37 ° C para la detección de colonias que probablemente contienen el inserto y se ejecutan en un 1,2% en gel de agarosa de ADN como en el paso 1.6. NOTA: Después de la digestión, una ligadura de éxito debe resultados en dos grupos, uno para cada vector y el inserto que se corresponden con su respectivo peso molecular (Figura 1). </li><li> Enviar las posibles colonias para la secuenciación de ADN utilizando promotor T7 cebadores directo e inverso a secuenciadores comerciales. </li></ol><ol><li> Desde el resultado de la secuenciación, seleccionar una colonia que ha sido ligado con éxito y utilizar el plásmido de ADN para la transformación en una E. huésped de expresión coli. </li><li> Descongele E. cepa de expresión coli (BL21 (DE3) pLysS) en hielo. Mientras tanto, las placas de agar caliente que contiene ampicilina (25 mg / ml) y cloranfenicol (34 mg / ml) a RT. NOTA: El plásmido presente en bacterias pLysS cepa BL21 (DE3) pLysS contiene un gen de resistencia a cloranfenicol. El plásmido pLysS contiene un gen represor T7 que se expresa constitutivamente a limitar la expresión de la proteína con fugas AECM. El cloranfenicol es necesario seleccionar para las células de bacterias que contiene pLysS durante el cultivo. </li><li> Repita el paso 1,10 para obtener transformó E. células de E. coli que están listos para expresar las proteínas artificiales. Envolver las placas de agar en Parafilm y almacenar al revés en 4 6; C durante un máximo de un mes. </li></ol> 3. Expresión bacteriana de AECM Proteínas <ol><li> Consulte la Figura 2, elegir una colonia de la placa de agar transformada con una punta de pipeta e inocular en 10 ml de caldo Terrific (TB) medios estériles (Tabla 1) que contiene tanto los antibióticos ampicilina y cloranfenicol en un tubo de ensayo. Incubar este cultivo iniciador a 37 ° CO / N (12.16 h) con agitación a 225 rpm. </li><li> Transferir 10 ml del cultivo iniciador en 1 L de medios de TB frescos y estériles suplementado con los mismos antibióticos en un matraz Erlenmeyer de 3 l. Incubar el cultivo a 37 ° C con agitación a 225 rpm durante 2-3 hr y observar la densidad óptica del cultivo (OD 600) alcanzado 0,6-0,8 pipeteando 1 ml de cultivo en una cubeta vacía para la lectura. Guardar 1 ml de cultivo antes de la inducción para la caracterización de SDS-PAGE. <ol><li> Para medir OD 600 del cultivo celular, preparar 1 vial de medios de comunicación de TB en una cubetapara una medición en blanco. Por otra parte, la transferencia de 1 ml de cultivo del matraz de cultivo en una nueva cubeta vacía. Medir la densidad óptica del cultivo usando un espectrofotómetro contra el blanco de control a una absorbancia de 600 nm. </li><li> Guardar la muestra (para el análisis SDS-PAGE posterior) mediante la transferencia de los 1 ml de muestra de cultivo en un tubo de microcentrífuga de 1,5 ml, se centrifuga a 12.000 xg durante 2 min, y decantar el sobrenadante. </li></ol></li><li> Inducir la cultura con isopropil β-D-1-tiogalactopiranósido (IPTG) a una concentración final de 1 mM y se incuba a 37 ° C con agitación a 225 rpm durante otras 4 h. Guardar 1 ml de cultivo al final de la inducción a 4 hr para la caracterización de SDS-PAGE. </li><li> Recoger las células mediante la transferencia de la cultura a botellas de centrífuga de 1 L y se centrifuga a 12.000 xg durante 30 min a 4 ° C. Descartar el sobrenadante, sopesar los sedimentos de células y resuspender en tampón TEN (Tris 1 M, 0,01 M EDTA, 0,1 M NaCl, pH = 8,0) a 0,5 g / ml. </li></ol> 4. La lisis de cultivos bacterianos <ol><li> Congelar la re-suspendido de cultivo celular a -80 ° CO / N. Descongelar el cultivo celular congelado en un baño de agua a temperatura ambiente o en hielo para lisar las células. Añadir 10 g / ml Deoxyribonuclease I (DNasa I), 10 mg / ml de ribonucleasa A (RNasa I), y 50 mg / ml de fluoruro de fenilmetilsulfonilo (PMSF) mientras que la descongelación y homogeneizar la solución con agitación lenta. </li><li> Después de todas las células resuspendidas se descongelan, ajustar la solución a pH 9,0 para aumentar la solubilidad de la proteína en agua 12. Añadir 6 N de NaOH gota a gota con agitación en hielo para alcanzar una consistencia homogénea. Lyse por la interrupción de ultrasonidos durante 20 min en hielo utilizando una punta plana de diámetro 2 mm, 5 pulso seg. </li><li> Centrifugar la solución de células a 12.000 xg durante 30 min a 4 ° C. Transferir el sobrenadante a un frasco vacío y limpio y se almacena a 4 ° C para la purificación posterior. </li><li> Mientras tanto, volver a suspender el sedimento celular nuevo con tampón TEN y volver a congelar a -80 ° C. Alisis celular completa, repetición de congelación / descongelación y sonicación proceso hasta tres veces. Ahorra un 20 lisado celular l para la caracterización de SDS-PAGE. </li></ol> 5. Purificación de proteínas usando AECM Inverse Transición Cycling <ol><li> Intercalar el lisado celular de la etapa 4.3 y proceder a purificar las proteínas AECM utilizando ITC, similar a la utilizada para la purificación de proteínas basado en elastina 21. Ciclos se hará con las diferentes temperaturas que son 4 ° C (referidos como &quot;frío&quot;) y 37 ° C (referidos como &quot;calientes&quot;) ciclos respectivamente. </li><li> Dividir el lisado celular en 50 ml botellas de centrífuga, y se centrifuga a 40.000 xg durante 2 horas a 4 ° C. El aspecto del sedimento celular debe ser de color marrón oscuro y buscar nasal. </li><li> Eliminar el sobrenadante con la pipeta para conseguir una separación limpia del sedimento. Recoger el sobrenadante en botellas de centrífuga limpio y añadir NaCl a una concentración final de 1 M (hasta un máximo de 3 M). Calientela solución a 37 ° C durante 2 horas con agitación a 225 rpm. NOTA: La adición de NaCl se disparará la transición del componente elastina de la AECM causando agregación. El sobrenadante se volverá turbia. Si la concentración de proteína es lo suficientemente alta, la proteína blanco espumoso se puede ver en los lados de la botella de centrífuga. </li><li> Centrifugar el sobrenadante del paso 5.3 a 40.000 xg durante 2 horas a 37 ° C. Decantar el sobrenadante. Triturar el sedimento usando una espátula de metal y resuspender los bits de pellets en autoclave de agua destilada enfriada con hielo (50 mg / ml) con agitación vigorosa O / N a 4 ° C utilizando una barra de agitación magnética y la placa. El sedimento debe ser completamente disuelto. </li><li> Repita los pasos 5.2 a 5.4 durante otros tres a cinco veces para obtener una mayor pureza de la proteína. </li><li> Después del ciclo final de la purificación, desalar la solución de proteína por diálisis contra agua destilada a 4 ° C. Dializar solución de proteína contra el agua durante 2-3 días con los cambios de agua cada 4hr o 8 horas de O / N. Ahorre 20 l de la proteína purificada por SDS-PAGE y liofilizar el resto de la proteína purificada y almacenar a -80 ° C hasta su uso posterior. </li></ol> 6. Caracterización de AECM proteínas utilizando SDS-PAGE electroforesis <ol><li> Preparar un gel de SDS-PAGE al 12% (si el peso molecular es grande, utilice 8% en geles de SDS-PAGE para una mejor separación). Añadir tampón de carga SDS 2x a cada muestra, calentar las muestras durante 10 min a 100 ° C y ejecutar muestras en el gel durante 1 hora a 100 V o hasta que la escalera de proteína correspondiente al peso molecular de la proteína AECM alcanza el medio de la gel. </li><li> Recuperar el gel de la SDS-PAGE configurar y añadir Coomassie Brilliant mancha azul (Tabla 2) con un volumen de sumergir el gel durante 1 hora en una placa de Petri. Enjuague el gel en una solución decolorante (Tabla 2) durante 5 min en un eje de balancín. Cambie la solución decolorante, y continuar destain el gel con balanceo hasta que el fondo de tél gel se vuelve claro. Las bandas de proteínas deben ser claramente visibles. </li><li> Comparación de la posición de la proteína diana contra la escalera de proteínas para determinar el peso molecular de la proteína diana. </li><li> Para confirmar aún más la presencia de la proteína diana, lleve a cabo el Western Blot como en el paso 7. </li></ol> 7. Caracterización de AECM proteínas usando Western Blotting <ol><li> Ejecute las muestras en un gel SDS-PAGE al 12%, como en la sección 6 sin manchas. </li><li> Cortar membrana de nitrocelulosa a un tamaño ligeramente mayor que el gel SDS-PAGE. Cortar 4 trozos de papel de filtro para el mismo tamaño. </li><li> Moje un pedazo de papel de filtro con transferencia Western Buffer (20% v / v de metanol, 25 mM Tris, 190 mM de glicina, pH 8,3), colocar el gel SDS-PAGE en la parte superior del papel de filtro, seguido de otro pedazo de papel de filtro , a continuación, la membrana de nitrocelulosa y una final otros dos trozos de papel de filtro. Asegúrese de que el conjunto de configuración se sumerge con tampón de transferencia occidental suficiente. NOTA: El gel y la membrana no deben estar en contacto en este punto de tiempo como proteínas podrían unirse a la membrana tras el contacto. </li><li> Transferir las proteínas en la membrana de nitrocelulosa mediante la colocación de dos papeles de filtro en una unidad de transferencia semiseca occidental, seguido de la membrana de nitrocelulosa, y coloque cuidadosamente el gel SDS-PAGE de dentro y de la membrana, por último colocar el último papel de filtro húmedo en dos el gel SDS-PAGE. Ejecutar la transferencia oeste a 45 mA, 30 min. Añadir búfer si el voltaje es superior a 30 V. NOTA: De preferencia colocar el gel SDS-PAGE en la membrana con un intento y evitar cambiar el gel de forma innecesaria en la membrana como proteínas podrían unirse a la membrana al entrar en contacto. </li><li> Recuperar y bloquear la membrana de nitrocelulosa con solución de bloqueo (leche desnatada al 5% en PBS pH 7,4, se filtró con papel de filtro) durante 2 horas a RT. Deshágase de tampón de bloqueo y añadir PBS para enjuagar. NOTA: El cambio a los nuevos guantes para evitar la contaminación de la membrana con prote no deseadoins. </li><li> Incubar anticuerpo anti-His primaria con la dilución en PBS a 1: 1000 a RT durante 1 hr con balanceo. Preparar una mezcla en un volumen que podría sumergir toda la membrana, por ejemplo, con relación de dilución 1: 1.000, añadir 1 l de anticuerpo (concentración Stock: 1 mg / ml) a 999 l de PBS para una mezcla total de 1 ml. </li><li> Enjuague la membrana con 5 ml de PBST (PBS con 0,1% de Tween 20). </li><li> Incubar con el anticuerpo secundario conjugado con peroxidasa de rábano picante (HRP) con una dilución en PBS a 1: 5000 a RT durante 1 hr con balanceo. Preparar una mezcla en un volumen que podría sumergir toda la membrana, por ejemplo, con relación de dilución 1: 5.000, añadir 1 l de anticuerpo (concentración Stock: 1 mg / ml) a 4999 l de PBS para una mezcla total de 5 ml. </li><li> Se lava la membrana con 5 ml de PBST y repetir dos veces. </li><li> Aplicar el sustrato quimioluminiscente a la membrana con un volumen para cubrir la membrana y se incuba siguiendo las instrucciones para el sustrato utilizado. NOTA: La sensibilidad de la detección de la proteína sustrato que puede variar, se recomienda un sustrato con la máxima sensibilidad para señales muy bajas en el caso de aumento de la concentración de anticuerpo no aumenta señales. </li><li> Captura de las señales de quimioluminiscencia utilizando un generador de imágenes basada cámara CCD. Ajustar la relación de dilución de anticuerpo primario y secundario si las señales son débiles y no específica. Incubar ya en solución de bloqueo si la señal de fondo es alto. </li></ol>

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	<li>Chen, Q., Liang, S., Thouas, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastomeric+biomaterials+for+tissue+engineering.">Elastomeric biomaterials for tissue engineering.</a> Prog Polym Sci. 38 (3-4), 584-671 (2013).</li><li>Groeber, F., Holeiter, M., Hampel, M., Hinderer, S., Schenke-Layland, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+tissue+engineering+-+In+vivo+and+in+vitro+applications.">Skin tissue engineering - In vivo and in vitro applications.</a> Adv Drug Deliv Rev. 63 (4-5), 352-366 (2011).</li><li>Kushner, A. M., Guan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modular+Design+in+Natural+and+Biomimetic+Soft+Materials.">Modular Design in Natural and Biomimetic Soft Materials.</a> Angewandte Chemie International Edition. 50 (39), 9026-9057 (2011).</li><li>Gagner, J. E., Kim, W., Chaikof, E. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+protein-based+biomaterials+for+medical+applications.">Designing protein-based biomaterials for medical applications.</a> Acta Biomater. 10 (4), 1542-1557 (2014).</li><li>Gomes, S., Leonor, I. B., Mano, J. F., Reis, R. 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=Natural+and+genetically+engineered+proteins+for+tissue+engineering.">Natural and genetically engineered proteins for tissue engineering.</a> Prog Polym Sci. 37 (1), 1-17 (2012).</li><li>Werkmeister, J. A., Ramshaw, J. A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recombinant+protein+scaffolds+for+tissue+engineering.">Recombinant protein scaffolds for tissue engineering.</a> Biomedical Materials. 7 (1), 012002 (2012).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomaterials+for+tissue+engineering+of+skin.">Biomaterials for tissue engineering of skin.</a> Materials Today. 11 (5), 26-35 (2008).</li><li>Fong, E., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+Cell+Migration+on+Artificial+Extracellular+Matrix+Proteins+Containing+Full-Length+Fibronectin+Domains.">Collective Cell Migration on Artificial Extracellular Matrix Proteins Containing Full-Length Fibronectin Domains.</a> Advanced Materials. 22 (46), 5271-5275 (2010).</li><li>Ratner, B. D., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BIOMATERIALS:+Where+We+Have+Been+and+Where+We+are+Going.">BIOMATERIALS: Where We Have Been and Where We are Going.</a> Annual Review of Biomedical Engineering. 6 (1), 41-75 (2004).</li><li>Cai, L., Heilshorn, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+ECM-mimetic+materials+using+protein+engineering.">Designing ECM-mimetic materials using protein engineering.</a> Acta Biomater. 10 (4), 1751-1760 (2014).</li><li>Annabi, N., 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=Elastomeric+recombinant+protein-based+biomaterials.">Elastomeric recombinant protein-based biomaterials.</a> Biochemical Engineering Journal. 77, 110-118 (2013).</li><li>Heilshorn, S. C., DiZio, K. A., Welsh, E. R., Tirrell, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endothelial+cell+adhesion+to+the+fibronectin+CS5+domain+in+artificial+extracellular+matrix+proteins.">Endothelial cell adhesion to the fibronectin CS5 domain in artificial extracellular matrix proteins.</a> Biomaterials. 24 (23), 4245-4252 (2003).</li><li>Simnick, A. J., Lim, D. W., Chow, D., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biomedical+and+Biotechnological+Applications+of+Elastin-Like+Polypeptides.">Biomedical and Biotechnological Applications of Elastin-Like Polypeptides.</a> Polymer Reviews. 47 (1), 121-154 (2007).</li><li>Tjin, M. S., Chua, A. W. C., Ma, D. R., Lee, S. T., Fong, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+Epidermal+Keratinocyte+Cell+Response+on+Integrin-Specific+Artificial+Extracellular+Matrix+Proteins.">Human Epidermal Keratinocyte Cell Response on Integrin-Specific Artificial Extracellular Matrix Proteins.</a> Macromolecular Bioscience. 14 (8), 1125-1134 (2014).</li><li>MacNeil, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progress+and+opportunities+for+tissue-engineered+skin.">Progress and opportunities for tissue-engineered skin.</a> Nature. 445 (7130), 874-880 (2007).</li><li>Kariya, Y., 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=Differential+regulation+of+cellular+adhesion+and+migration+by+recombinant+laminin-5+forms+with+partial+deletion+or+mutation+within+the+G3+domain+of+&#945;3+chain.">Differential regulation of cellular adhesion and migration by recombinant laminin-5 forms with partial deletion or mutation within the G3 domain of &#945;3 chain.</a> J Cell Biochem. 88 (3), 506-520 (2003).</li><li>Shang, M., Koshikawa, N., Schenk, S., Quaranta, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+LG3+module+of+laminin-5+harbors+a+binding+site+for+integrin+&#945;3&#914;1+that+promotes+cell+adhesion,+spreading,+and+migration.">The LG3 module of laminin-5 harbors a binding site for integrin &#945;3&#914;1 that promotes cell adhesion, spreading, and migration.</a> J Biol Chem. 276 (35), 33045-33053 (2001).</li><li>Hassouneh, W., Christensen, T., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elastin-like+polypeptides+as+a+purification+tag+for+recombinant+proteins.">Elastin-like polypeptides as a purification tag for recombinant proteins.</a> Current Protocols in Protein Science. , 6.11.1-6.11.16 (2010).</li><li>Keeley, F., Mecham, R., Keeley, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ch.+4.">Ch. 4.</a> Evolution of Extracellular Matrix.Biology of Extracellular Matrix. , 73-119 (2013).</li><li>Le, D. 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=Self-Assembly+of+Elastin-Mimetic+Double+Hydrophobic+Polypeptides.">Self-Assembly of Elastin-Mimetic Double Hydrophobic Polypeptides.</a> Biomacromolecules. 14 (4), 1028-1034 (2013).</li><li>MacEwan, S. R., Hassouneh, W., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-chromatographic+Purification+of+Recombinant+Elastin-like+Polypeptides+and+their+Fusions+with+Peptides+and+Proteins+from+Escherichia+coli.">Non-chromatographic Purification of Recombinant Elastin-like Polypeptides and their Fusions with Peptides and Proteins from Escherichia coli.</a> Journal of visualized experiments: JoVE. (88), e51583 (2014).</li><li>Meyer, D. E., Chilkoti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetically+encoded+synthesis+of+protein-based+polymers+with+precisely+specified+molecular+weight+and+sequence+by+recursive+directional+ligation:+examples+from+the+elastin-like+polypeptide+system.">Genetically encoded synthesis of protein-based polymers with precisely specified molecular weight and sequence by recursive directional ligation: examples from the elastin-like polypeptide system.</a> Biomacromolecules. 3 (2), 357-367 (2002).</li></ol>

Los autores declaran que no tienen intereses financieros en competencia.

Ingeniería de proteínas recombinantes es una técnica versátil para crear materiales de proteínas novedosas utilizando un enfoque de abajo hacia arriba. Los materiales basados ​​en proteínas pueden ser diseñados para tener múltiples funcionalidades, adaptado de acuerdo con la aplicación de interés. Debido a la creciente avance en las tecnologías de clonación y expresión de proteínas, se ha convertido en relativamente simple (y rentable) para crear una variedad de proteínas artificiales de una manera re...

For several decades, both synthetic and natural materials have been explored for use as scaffolds in tissue engineering1,2. While synthetic materials such as polymers offer excellent structural integrity and tunable mechanical properties, they often have insufficient bioactivity to promote growth and infiltration of tissues. On the other hand, natural materials such as extracellular matrix (ECM) proteins have excellent biological activity, but have limitations such as batch-to-batch variability, rapid degradation and immunogenicity issues. As such, recombinant proteins are desired, since they can be designed to mimic only the desirable properties of native proteins3,4.
Recombinant protein engineering has garnered widespread interests as a versatile platform for the design and production of novel artificial protein biopolymers. By controlling the genetic sequence, the functionalities of the artificial proteins can be tailored for a wide variety of applications5,6. In particular, artificial extracellular matrix (aECM) proteins can be tailored to have multiple functionalities for applications in tissue engineering, regeneration and wound repair2,7. More importantly, advances in cloning and purification technologies have increased scalability and reduced the cost of manufacturing recombinant proteins tremendously. It is possible to produce large quantities of recombinant proteins at low production costs which are economic for use in the clinic5. 
Artificial extracellular matrix proteins have been developed for tissue engineering applications8-11. For instance, Tirrell et al. designed a small diameter vascular graft using artificial proteins containing fibronectin CS5 sequence and elastin-like repeats (ELP-CS5). They showed that human umbilical vein endothelial cells (HUVECs) were able to adhere and grow on these materials12. Others have also incorporated short bioactive sequences taken from fibronectin, collagen, laminin, fibrinogen and vitronectin as well as structural domains that mimic elastin, spider silk and collagens to create a variety of fusion proteins10. Bulk cross-linked films made out of elastin-based aECM proteins also exhibited mechanical properties similar to that of native elastin (elastic moduli ranges between 0.3-0.6 MPa)13. Subsequently, aECM proteins containing longer fibronectin fragments were also reported to accelerate wound healing in vitro due to increased integrin binding affinities8. 
Recently, integrin-specific artificial ECM proteins have been developed by Tjin and coworkers14. Each aECM protein contains a bioactive cell-binding domain taken from ECM components of native human skin2,7,15, such as laminin-5, collagen-IV and fibronectin. For example, the integrin &#945;3&#914;1 has been shown to bind the PPFLMLLKGSTR sequence found in the laminin-5 alpha-3 chain globular domain 3 (LG3)16,17. In their report, they showed that primary human skin epidermal keratinocytes preferentially engage different integrins for binding to each of the aECM proteins, depending on the type of cell binding domain present.
The aECM proteins discussed in the work by Tjin et al. contain flanking elastin-like domains {(VPGIG)2VPGKG(VPGIG)2}8 that confer elasticity which mimics the mechanical properties of human skin. In addition, the incorporation of lysine residues within the elastin-like repeats also increases the overall protein solubility in aqueous solvents. In addition, the lysine residues also serve as crosslinking sites to facilitate the formation of crosslinked aECM films12. Inclusion of elastin-like repeats within the aECM protein sequence allow the proteins to be readily purified via Inverse Transition Cycling (ITC)14. Elastins undergo a sharp and reversible phase transition at a specific temperature known as the lower critical solution temperature (LCST) or the inverse transition temperature (Tt)18-20. Elastins and elastin-like repeats adopt hydrophilic random coil conformations below their LCST and become soluble in water, whereas above their LCST, elastins aggregate rapidly into micron-size particles. Such phase transitions are reversible and hence, can be exploited to allow elastin-based aECM proteins to be readily purified via the ITC technique21.
In this work, we report a generalized procedure to design, construct and purify artificial ECM proteins containing bioactive cell-binding domains, fused to elastin-like repeats. The process to design and clone the plasmids that encode for the amino acid sequences for the aECM proteins is described. The steps involved to purify the aECM proteins using ITC are outlined. Finally, the methods to determine the purity of the aECM proteins obtained using SDS-PAGE electrophoresis and Western Blotting are discussed.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>pET22b (+)</td>
			<td>Novagen</td>
			<td>69744</td>
			<td>T7 expression vectors with resistance to ampicillin&#160;</td>
		</tr>
		<tr>
			<td>BL21(DE3)pLysS&#160;</td>
			<td>Invitrogen</td>
			<td>C6060-03</td>
			<td>additional antibiotics - chloramphenicol</td>
		</tr>
		<tr>
			<td>Isopropyl-beta-D-thiogalactoside (IPTG)</td>
			<td>Gold Biotechnology</td>
			<td>I2481C</td>
			<td>1M stock solution with autoclaved water, make fresh prior to induction.</td>
		</tr>
		<tr>
			<td>QIAprep Spin Miniprep Kit</td>
			<td>Qiagen</td>
			<td>27106</td>
			<td>plasmid isolation kit</td>
		</tr>
		<tr>
			<td>T4 ligase</td>
			<td>New England Biolabs</td>
			<td>M0202S</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin</td>
			<td>Affymetrix</td>
			<td>11259</td>
			<td></td>
		</tr>
		<tr>
			<td>Chloramphenicol</td>
			<td>Affymetrix</td>
			<td>23660</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymoclean&#8482; gel DNA recovery kit</td>
			<td>Zymo Research</td>
			<td>D4001</td>
			<td></td>
		</tr>
		<tr>
			<td>XL10-gold strain</td>
			<td>Agilent Technologies</td>
			<td>200315</td>
			<td></td>
		</tr>
	</tbody>
</table>

design and construction of artificial extracellular matrix (aecm) proteins from escherichia coli for skin tissue engineering

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins that have incorporated functional domains derived from nature (or created de novo) have been reported. In particular, artificial extracellular matrix (aECM) proteins have been developed; these aECM proteins consist of biological domains taken from fibronectin, laminins and collagens and are combined with structural domains including elastin-like repeats, silk and collagen repeats. To date, aECM proteins have been widely investigated for applications in tissue engineering and wound repair. Recently, Tjin and coworkers developed integrin-specific aECM proteins designed for promoting human skin keratinocyte attachment and propagation. In their work, the aECM proteins incorporate cell binding domains taken from fibronectin, laminin-5 and collagen IV, as well as flanking elastin-like repeats. They demonstrated that the aECM proteins developed in their work were promising candidates for use as substrates in artificial skin. Here, we outline the design and construction of such aECM proteins as well as their purification process using the thermo-responsive characteristics of elastin.

En el diseño de proteínas de fusión que contienen repeticiones tipo elastina, es importante para mantener un contenido de elastina en general, gran parte suficiente de la proteína de fusión 18. Esto es para asegurar que el constructo de proteína de fusión conserva sus características tipo elastina, con el fin de utilizar ITC para la purificación. El diseño y las secuencias de proteínas descritas en esta sección AECM se tomaron específicamente de la obra por Tjin et al. 14. En ...

Watch this Scientific Journal Video about Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering at JoVE.com

Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats.

Diseño y Construcción de Artificial matriz extracelular (AECM) Las proteínas de<em&gt; Escherichia coli</em&gt; Para la piel Ingeniería de Tejidos

Recombinant technology is a versatile platform to create novel artificial proteins with tunable properties. For the last decade, many artificial proteins ...

design-construction-artificial-extracellular-matrix-aecm-proteins

Research

JoVE Journal

Bioengineering

Design and Construction of Artificial Extracellular Matrix (aECM) Proteins from Escherichia coli for Skin Tissue Engineering

8.8K vistas.  Nanyang Technological University. Recombinant technologies have enabled material designers to create novel artificial proteins with customized functionalities for tissue engineering applications. For example, artificial extracellular matrix proteins can be designed to incorporate structural and biological domains derived from native ECMs. Here, we describe the construction and purification of aECM proteins containing elastin-like repeats. 

Video: Design and Construction of Artificial Extracellular Matrix aECM Proteins from Escherichia coli for Skin Tissue Engineering

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

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

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

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

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

Investigación

Bioingeniería

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.

We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.

Diseño de un Carga mecánica Biaxial biorreactor para Ingeniería de Tejidos

We  designed a loading device that is capable of applying uniaxial or biaxial  mechanical strain to a tissue engineered biocomposites fabricated for  ...

Tissue Engineering: Construction of a Multicellular 3D Scaffold for the Delivery of Layered Cell Sheets

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to assist the body in recovery and repair. For example, TE approaches may be able to attenuate heart remodeling after myocardial infarction (MI) and possibly increase total heart function to a near normal pre-MI level.4 As with any functional tissue, successful regeneration of cardiac tissue involves the proper delivery of multiple cell types with environmental cues favoring integration and survival of the implanted cell/tissue graft. Engineered tissues should address multiple parameters including: soluble signals, cell-to-cell interactions, and matrix materials evaluated as delivery vehicles, their effects on cell survival, material strength, and facilitation of cell-to-tissue organization. Studies employing the direct injection of graft cells only ignore these essential elements.2,5,6 A tissue design combining these ingredients has yet to be developed. Here, we present an example of integrated designs using layering of patterned cell sheets with two distinct types of biological-derived materials containing the target organ cell type and endothelial cells for enhancing new vessels formation in the &#8220;tissue&#8221;. Although these studies focus on the generation of heart-like tissue, this tissue design can be applied to many organs other than heart with minimal design and material changes, and is meant to be an off-the-shelf product for regenerative therapies. The protocol contains five detailed steps. A temperature sensitive Poly(N-isopropylacrylamide) (pNIPAAM) is used to coat tissue culture dishes. Then, tissue specific cells are cultured on the surface of the coated plates/micropattern surfaces to form cell sheets with strong lateral adhesions. Thirdly, a base matrix is created for the tissue by combining porous matrix with neovascular permissive hydrogels and endothelial cells. Finally, the cell sheets are lifted from the pNIPAAM coated dishes and transferred to the base element, making the complete construct.

For creation of highly organized structures of complex tissue, one must assemble multiple material and cell types into an integrated composite. This combinatorial design incorporates organ-specific layered cell sheets with two distinct biologically-derived materials containing a strong fibrous matrix base, and endothelial cells for enhancing new vessels formation.

Ingeniería de tejidos: Construcción de un multicelular 3D Andamios para la entrega de láminas de células en capas

Many tissues, such as the adult human hearts, are unable to adequately regenerate after damage.2,3 Strategies in tissue engineering propose innovations to ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Epithelial Cell Repopulation and Preparation of Rodent Extracellular Matrix Scaffolds for Renal Tissue Development

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model substrates for organ-scale tissue development. Sprague Dawley rat kidneys are cannulated by inserting a catheter into the renal artery and perfused with a series of low-concentration detergents (Triton X-100 and sodium dodecyl sulfate (SDS)) over 26 hr to derive intact, whole-kidney scaffolds with intact perfusable vasculature, glomeruli, and renal tubules. Following decellularization, the renal scaffold is placed inside a custom-designed perfusion bioreactor vessel, and the catheterized renal artery is connected to a perfusion circuit consisting of: a peristaltic pump; tubing; and optional probes for pH, dissolved oxygen, and pressure. After sterilizing the scaffold with peracetic acid and ethanol, and balancing the pH (7.4), the kidney scaffold is prepared for seeding via perfusion of culture medium within a large-capacity incubator maintained at 37 &#176;C and 5% CO2. Forty million renal cortical tubular epithelial (RCTE) cells are injected through the renal artery, and rapidly perfused through the scaffold under high flow (25 ml/min) and pressure (~230 mmHg) for 15 min before reducing the flow to a physiological rate (4 ml/min). RCTE cells primarily populate the tubular ECM niche within the renal cortex, proliferate, and form tubular epithelial structures over seven days of perfusion culture. A 44 &#181;M resazurin solution in culture medium is perfused through the kidney for 1 hr during medium exchanges to provide a fluorometric, redox-based metabolic assessment of cell viability and proliferation during tubulogenesis. The kidney perfusion bioreactor permits non-invasive sampling of medium for biochemical assessment, and multiple inlet ports allow alternative retrograde seeding through the renal vein or ureter. These protocols can be used to recellularize kidney scaffolds with a variety of cell types, including vascular endothelial, tubular epithelial, and stromal fibroblasts, for rapid evaluation within this system.

This protocol describes decellularization of Sprague Dawley rat kidneys by antegrade perfusion of detergents through the vasculature, producing acellular renal extracellular matrices that serve as templates for repopulation with human renal epithelial cells. Recellularization and use of the resazurin perfusion assay to monitor growth is performed within specially-designed perfusion bioreactors.

La repoblación de las células epiteliales y Preparación de roedores matriz extracelular andamios para el Desarrollo del tejido renal

This protocol details the generation of acellular, yet biofunctional, renal extracellular matrix (ECM) scaffolds that are useful as small-scale model ...

Engineering Three-dimensional Epithelial Tissues Embedded within Extracellular Matrix

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, which is regulated by a variety of soluble and physical signals in the microenvironment. Described here is a method created to study the process of branching morphogenesis by forming engineered three-dimensional (3D) epithelial tissues of defined shape and size that are completely embedded within an extracellular matrix (ECM). This method enables the formation of arrays of identical tissues and enables the control of a variety of environmental factors, including tissue geometry, spacing, and ECM composition. This method can also be combined with widely used techniques such as traction force microscopy (TFM) to gain more information about the interactions between cells and their surrounding ECM. The protocol can be used to investigate a variety of cell and tissue processes beyond branching morphogenesis, including cancer invasion.

This manuscript describes a soft lithography-based technique to engineer uniform arrays of three-dimensional (3D) epithelial tissues of defined geometry surrounded by extracellular matrix. This method is amenable to a wide variety of cell types and experimental contexts and allows for high-throughput screening of identical replicates.

Ingeniería tridimensionales epiteliales tejidos embebidos dentro de la matriz extracelular

The architecture of branched organs such as the lungs, kidneys, and mammary glands arises through the developmental process of branching morphogenesis, ...

Tunable Hydrogels from Pulmonary Extracellular Matrix for 3D Cell Culture

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung tissue from porcine, rat, or mouse, the tissue is perfused and submerged in subsequent chemical detergents to remove the cellular debris. Histological comparison of the tissue before and after processing confirms removal of over 95% of double stranded DNA and alpha galactosidase staining suggests the majority of cellular debris is removed. After decellularization, the tissue is lyophilized and then cryomilled into a powder. The matrix powder is digested for 48 hr in an acidic pepsin digestion solution and then neutralized to form the pregel solution. Gelation of the pregel solution can be induced by incubation at 37 &#176;C and can be used immediately following neutralization or stored at 4 &#176;C for up to two weeks. Coatings can be formed using the pregel solution on a non-treated plate for cell attachment. Cells can be suspended in the pregel prior to self-assembly to achieve a 3D culture, plated on the surface of a formed gel from which the cells can migrate through the scaffold, or plated on the coatings. Alterations to the strategy presented can impact gelation temperature, strength, or protein fragment sizes. Beyond hydrogel formation, the hydrogel stiffness may be increased using genipin.

This is a method to create a 3-dimensional cell culture scaffold from pulmonary extracellular matrix. Intact lung is processed into hydrogels that can support the growth of cells in three-dimensions.

Los hidrogeles sintonizable de la matriz extracelular pulmonar por la 3D de Cultivos Celulares

Here we present a method for establishing multiple component cell culture hydrogels for in vitro lung cell culture. Beginning with healthy en bloc lung ...

Fabrication of Extracellular Matrix-derived Foams and Microcarriers as Tissue-specific Cell Culture and Delivery Platforms

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus of this article is on the methods for fabricating ECM-derived porous foams and microcarriers for use as biologically-relevant substrates in advanced 3D in vitro cell culture models or as pro-regenerative scaffolds and cell delivery systems for tissue engineering and regenerative medicine. Using decellularized tissues or purified insoluble collagen as a starting material, the techniques can be applied to synthesize a broad array of tissue-specific bioscaffolds with customizable geometries. The approach involves mechanical processing and mild enzymatic digestion to yield an ECM suspension that is used to fabricate the three-dimensional foams or microcarriers through controlled freezing and lyophilization procedures. These pure ECM-derived scaffolds are highly porous, yet stable without the need for chemical crosslinking agents or other additives that may negatively impact cell function. The scaffold properties can be tuned to some extent by varying factors such as the ECM suspension concentration, mechanical processing methods, or synthesis conditions. In general, the scaffolds are robust and easy to handle, and can be processed as tissues for most standard biological assays, providing a versatile and user-friendly 3D cell culture platform that mimics the native ECM composition. Overall, these straightforward methods for fabricating customized ECM-derived foams and microcarriers may be of interest to both biologists and biomedical engineers as tissue-specific cell-instructive platforms for in vitro and in vivo applications.

The tissue-specific extracellular matrix (ECM) is a key mediator of cell function. This article describes methods for synthesizing pure ECM-derived foams and microcarriers that are stable in culture without the need for chemical crosslinking for applications in advanced 3D in vitro cell culture models or as pro-regenerative bioscaffolds.

La fabricación de espumas y microportadores derivados de la matriz extracelular como específica de tejido de cultivo celular y las plataformas de entrega

Cell function is mediated by interactions with the extracellular matrix (ECM), which has complex tissue-specific composition and architecture. The focus ...

Fabricating a Kidney Cortex Extracellular Matrix-Derived Hydrogel

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust mechanical support for in vitro cell culture but lack the necessary protein and ligand composition to elicit physiological behavior from cells. This manuscript describes a fabrication method for a kidney cortex ECM-derived hydrogel with proper mechanical robustness and supportive biochemical composition. The hydrogel is fabricated by mechanically homogenizing and solubilizing decellularized human kidney cortex ECM. The matrix preserves native kidney cortex ECM protein ratios while also enabling gelation to physiological mechanical stiffnesses. The hydrogel serves as a substrate upon which kidney cortex-derived cells can be maintained under physiological conditions. Furthermore, the hydrogel composition can be manipulated to model a diseased environment which enables the future study of kidney diseases.

Here we present a protocol to fabricate a kidney cortex extracellular matrix-derived hydrogel to retain the native kidney extracellular matrix (ECM) structural and biochemical composition. The fabrication process and its applications are described. Finally, a perspective on using this hydrogel to support kidney-specific cellular and tissue regeneration and bioengineering is discussed.

Fabricación de un hidrogel de derivados de matriz extracelular renal corteza

Extracellular matrix (ECM) provides important biophysical and biochemical cues to maintain tissue homeostasis. Current synthetic hydrogels offer robust ...

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

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

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

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

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