Este trabajo cuenta con el apoyo financiero de NIH (Becas 1R01AR072027-01, 1R03AR069383-01), NSF Career Award (1653702) y Universidad de Connecticut.

1. Síntesis de JBNT
NOTA: El monómero JBNT se preparó como se publicó anteriormente11.
<ol><li>Síntesis del compuesto A1<ol>
<li>Preparar una solución que contenga 8,50 g de ácido cianoacético de 2 y 9,80 g de etilcarbamato en 25 ml de tolueno y 2,5 ml de N, N-dimetilformamida. Añadir 4,90 ml de cloruro de fósforo en sentido dropwise. A continuación, caliente la mezcla a 70 °C y siga revolviendo durante 1,5 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y vierta 100 g de agua helada. Extraiga la capa acuosa con acetato de etilo (3 x 250 ml) y lave con 100 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore volátiles bajo vacío para obtener un sólido de color amarillo pálido.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (3% metanol/diclorometano) para producir 15,61 g del compuesto A1 como polvo blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A2<ol>
<li>Mezclar 3,12 g del compuesto A1 y 2,75 g de carbonato de potasio en 50 ml de N, N-dimetilformamida. Revuelva a temperatura ambiente durante 2 h.</li>
		<li>Añadir 2,6 ml de disulfuro de carbono a la suspensión de reacción. Sigue revolviendo durante 4 h.</li>
		<li>Añadir etanol absoluto a la mezcla de reacción a 0 °C. Filtre el precipitado amarillo hacia fuera y lave con éter de dietil. Secar bajo vacío durante la noche para producir 6,18 g del compuesto A2 como sólido de color amarillo pálido.</li>
	</ol></li>
	<li>Síntesis del compuesto A3<ol>
<li>Añadir 2,7 ml de yoduro de metilo en 15 ml de acetonitrilo. Además, preparar por separado una solución del compuesto A2 añadiendo 6,18 g de A2 a 80 ml de agua/acetonitrilo (relación 7:3). Mezcle las dos soluciones y revuelva a temperatura ambiente durante 30 minutos.</li>
		<li>Caliente la mezcla de reacción a 95 °C y siga revolviendo durante 3 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y luego evapore los volátiles bajo vacío.</li>
		<li>Extraiga el residuo 3x con 100 ml de acetato de etilo y lávelo con salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para obtener un sólido amarillo.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (30% acetato etílico/hexanes) para producir 4,52 g del compuesto A3 como sólido amarillo claro.</li>
	</ol></li>
	<li>Síntesis del compuesto A4<ol>
<li>Preparar solución de allylamina mediante la adición de 925 μL de allylamina en 20 ml de etanol. Añada esta solución gota a gota a la solución del compuesto A3, preparado añadiendo 3,14 g de A3 en 75 ml de etanol absoluto, más de 30 min. A continuación, caliente la mezcla de reacción para reflujo durante 16 h.</li>
		<li>Enfríe la mezcla de reacción a temperatura ambiente y evapore los volátiles para dar un sólido amarillo.</li>
		<li>Someta la cromatografía de columna flash de gel amarillo a sílice (50% acetato de etilo/hexanes al 2% de metanol/diclorometano) para producir 1,80 g del compuesto A4 como cristal blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A5<ol>
<li>Añadir 2,05 g de clorhidrato de guanidinio y 7,8 ml de etóxido de sodio (21% en etanol) en 14 ml de etanol absoluto. Caliente la mezcla a 45 °C durante 15 min.</li>
		<li>Filtre el material insoluble y agregue el filtrado directamente a la solución del compuesto A4, preparado añadiendo 2,70 g de A4 en etanol absoluto de 40 ml. Caliente la mezcla de reacción para reflujo durante 16 h.</li>
		<li>Filtre el precipitado hacia fuera para obtener 2,65 g del compuesto A5 como un sólido blanquecino.</li>
	</ol></li>
	<li>Síntesis del compuesto A6<ol>
<li>Añadir 24,9 ml de trietiltamina lentamente a la mezcla de purines obtenida añadiendo 2,11 g de compuesto A5 y 1,10 g de 4-Dimetilminopyridina en 120 ml de tetrahidrofurano durante 1 min. Revuelva a temperatura ambiente durante 2 minutos.</li>
		<li>Añadir 20,7 ml de di-tert-butyl dicarbonato gota a gota a la mezcla de reacción y seguir revolviendo a temperatura ambiente durante 40 h.</li>
		<li>Agregue 20 ml de agua para apagar la reacción. Evaporar los volátiles bajo vacío.</li>
		<li>Extraiga el residuo viscoso rojo con 500 ml de acetato de etilo. A continuación, lávalo con 250 ml de agua, 75 ml de ácido cítrico al 10%, 2 veces con 200 ml de agua, 200 ml de bicarbonato sódico saturado y 200 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo el vacío para dar un sólido rojo/naranja.</li>
		<li>Someta la cromatografía de columna flash de gel sólido a sílice (0-20% acetato de etilo/hexanes) para producir 3,87 g del compuesto A6 como espuma blanca.</li>
	</ol></li>
	<li>Síntesis del compuesto A7<ol>
<li>Añadir N-metilmorfina N-óxido (50 wt.% en agua, 1,64 ml) en sentido dropwise a la solución del compuesto A6 (2,93 g) en acetona / agua (8:1, 58,5 mL) y revolver a temperatura ambiente durante 5 minutos.</li>
		<li>Añadir tetraóxido de osmio (4% solución acuosa) a la mezcla durante un período de 3 minutos y seguir revolviendo a temperatura ambiente durante 24 h.</li>
		<li>Encienda la reacción con sulfito de sodio acuoso de 1,0 M hasta que la solución se vuelva incolora. Retire los volátiles bajo vacío para dar lugar a un purines blancos en agua. Extraiga el residuo con 350 ml de diclorometano y luego lave con 150 ml de agua seguido de un lavado con 150 ml de salmuera.</li>
		<li>Secar la capa orgánica sobre sulfato de sodio anhidro, filtrar y evaporar los volátiles bajo vacío para producir 2,45 g del compuesto A7 como un sólido blanco.</li>
	</ol></li>
	<li>Síntesis del compuesto A8<ol>
<li>Añadir 760 g de perioduro de sodio a la mezcla de 1,36 g del compuesto A7 en 35 ml de diclorometano/agua (6:1, 35 ml) y revuelva a temperatura ambiente durante 42 h.</li>
		<li>Filtre el material insoluble y concentre el filtrado bajo vacío. Extraiga el residuo resultante con 250 ml de diclorometano, y luego lave con 100 ml de agua y 100 ml de salmuera. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para dar el producto crudo.</li>
		<li>Someta el producto crudo a cromatografía de columna flash de gel de sílice (5-40% acetato de etilo/hexanes, luego 5% metanol/diclorometano) para producir 1,08 g del compuesto A8.</li>
	</ol></li>
	<li>Síntesis del compuesto A9<ol>
<li>Preparar una solución de 830 mg del compuesto A8 en 15 ml de 1,2-dicloroetano. Añadir la solución de gota a gota a la mezcla de 6-benzyloxycarbonylamino-2-L-amino-ácido hexanoico trimetilsilyl éster etílico (649.5 mg) y N,N-diisopropylethylamine (591 μL) en 24 ml de 1,2-dicloroetano durante 3 min y revuelve a temperatura ambiente durante 15 min.</li>
		<li>Añadir 360,3 mg de triacetoxyborohidrato sódico a la solución y seguir revolviendo a temperatura ambiente durante 24 h.</li>
		<li>Saciar la mezcla de reacción con 6 ml de agua. Extraer la mezcla de reacción 3x con 200 ml de diclorometano y lavar con 200 ml de ácido cítrico al 10% en agua, 2x con 200 ml de agua, 200 ml de bicarbonato sódico saturado y 200 ml de salmuera en ese orden. Seque la capa orgánica sobre sulfato de sodio anhidro, filtre y evapore los volátiles bajo vacío para obtener el producto crudo.</li>
		<li>Someta el producto crudo a cromatografía de columna flash de gel de sílice (5-30% acetato de etilo/hexanes) para producir 885 mg compuesto A9.</li>
	</ol></li>
	<li>Síntesis del monómero JBNT<ol>
<li>Disolver 0,54 g del compuesto A9 (0,54 g) en 10 ml de solución de ácido trifluoroacético/tioanisole al 94% y agitar a temperatura ambiente durante 72 h.</li>
		<li>Agregue el éter de dietil (80 ml) a la mezcla de reacción y centrífuga el precipitado hacia abajo. Vierta el ácido trifluoroacético claro que contiene sobrenadante y lave el precipitado blanco con éter de dietil y metanol para producir 168,6 mg de monómero crudo JBNT(Archivo Suplementario 1).</li>
		<li>Purifique el monómero JBNT crudo mediante una cromatografía líquida de alto rendimiento (HPLC) con una columna de fase inversa. El programa de aislamiento se muestra a continuación: Disolvente A: 100% agua; disolvente B: 100% acetonitrilo; disolvente C: solución de agua pH=1 HCl; caudal: 3 mL/min (véase la Tabla 1).</li>
		<li>Recoge el pico más grande a unos 7,2 minutos.</li>
	</ol></li>
</ol>2. Fabricación para JBNT/FN
<ol><li>Añadir 80 μL de solución acuosa JBNT de 1 mg/ml a 40 μL de solución acuosa FN de 1 mg/ml y pipeta varias veces.</li>
	<li>Compruebe la presencia de suspensión sólida blanca después de la tubería durante unos 10 s. NOTA: Se tomó un video para capturar el proceso de autoensamblaje de NM (Archivo suplementario 2).</li>
</ol>3. Observación de especímenes liofilizados
NOTA: Este paso se realiza para mostrar la estructura de andamios de NM hecha de JBNT y FN.
<ol><li>Prepare soluciones FN, JBNT y FN/JBNT NM para la observación de materiales liofilizados. Diluir 10 μL de 100 μg/ml de FN con 40 μL de agua destilada para hacer una solución de 20 μg/ml de FN.</li>
	<li>Preparar 100 mg/ml de solución de JBNT diluyendo 5 μL de 1 mg/ml de JBNTs en 45 μL de agua di.</li>
	<li>Mezclar 10 μL de 100 μg/mL FN y 5 μL de 1 mg/ml de JBNT en 35 μL de agua DI para formar la solución NM.</li>
	<li>Cubra tres pozos de microplacas inferiores redondas transparentes de 96 pozos con el FN, la solución JBNT y la solución NM, respectivamente. Cada pozo recibe 50 μL de la solución.</li>
	<li>Realizar la liofilización, como se describe a continuación, para visualizar la estructura del andamio del NM.<ol>
<li>Congele la placa a -80 °C durante la noche.</li>
		<li>Encienda el instrumento liofilizado y la bomba de vacío.</li>
		<li>Pulse el botón de enfriamiento para reducir la temperatura del sistema a -50 °C.</li>
		<li>Coloque la placa congelada en el instrumento liofilizado y cierre todas las aberturas de los instrumentos.</li>
		<li>Haga clic en el botón de inicio para reducir la presión del sistema a 0,9 mbar.</li>
		<li>Después de las 4 h, pulse el botón de aireación y abra la válvula para aumentar la presión del sistema a la presión atmosférica.</li>
		<li>Saque el plato del instrumento liofilizado.</li>
		<li>Utilice un microscopio para observar el FN, los JBNT y el NM autoensamblado resultante. Tome varias fotografías con una cámara para los materiales.</li>
	</ol>
</li>
</ol>4. Medición de espectros de absorción
NOTA: Utilice el cambio de espectros para FN y JBNT para presentar el JBNT/FN NM12autoensamblado.
<ol><li>Mezclar 30 μL de 100 μg/mL FN con 15 μL de agua destilada y 5 μL de 1 mg/ml de JBNT para preparar la solución JBNT/FN NM. La suspensión sólida blanca se puede ver después de canalizar la solución varias veces. NOTA: Las concentraciones finales de JBNT y FN en la solución son 100 μg/mL y 60 μg/mL, respectivamente. Las soluciones JBNT y FN a la misma concentración a la utilizada en la solución NM también se prepararon como soluciones de control.</li>
	<li>Diluir 5 μL de 1 mg/ml de JBNT con 45 μL de agua destilada para preparar la solución de control JBNT.</li>
	<li>Diluir 30 μL de 100 μg/mL FN con 20 μL de agua destilada para preparar la solución de control FN.</li>
	<li>Mida los espectros de absorción UV-vis de las soluciones FN, JBNT y JBNT/FN NM con el espectrofotómetro (ver Tabla de materiales)para caracterizar el ensamblaje de NM.<ol>
<li>Haga clic en el botón UV-Vis. Limpie la superficie de ensayo del espectrofotómetro y deje caer 2 μL de agua destilada sobre él. Mida como en blanco de 190-850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución FN en él. Mida los espectros de absorción UV-Vis de la solución FN de 190 nm a 850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución JBNT sobre ella. Mida los espectros de absorción de la solución JBNT de 190 nm a 850 nm.</li>
		<li>Limpie la superficie de prueba del espectrofotómetro y suelte 2 μL de solución JBNT/FN NM en él. Mida los espectros de absorción de la solución JBNT/FN NM de 190 nm a 850 nm.</li>
	</ol>
</li>
</ol>5. Preparación de JBNT/FN NM para microscopía electrónica de transmisión (TEM)
<ol><li>Limpie varias rejillas antes de manchar negativamente con un limpiador de plasma básico (consulte Tabla de materiales).</li>
	<li>Realice la tinción negativa de las muestras siguiendo dos procesos diferentes.<ol>
<li>Gota 3 μL de 1 mg/ml de solución acuosa JBNTs y 3 μL de solución JBNT/FN NM en rejillas separadas y déjela durante 2 minutos. A continuación, enjuague cada rejilla con 100 μL de solución de acetato de urilo (UA) del 0,5%. Escurrir el exceso de solución con papel filtrante y secar el aire de las rejillas.</li>
		<li>Mezcle 9 μL de solución JBNT/FN NM con 3 μL de solución 2.0% UA y pipeta varias veces. Pipetear la solución mixta en las rejillas y mantenerla en las rejillas durante 2 minutos. Utilice un papel de filtro para eliminar la solución mixta de las rejillas y secar las rejillas a temperatura ambiente.</li>
	</ol>
</li>
	<li>Por último, caracterizar la muestra de JBNT, JBNT/FN NM manchada con el paso 5.2.1, y JBNT/FN NM manchada con el paso 5.2.2 con el TEM.</li>
</ol>6. Ensayo de función biológica in vitro
<ol><li>Añada 200 μL de controles negativos (NC, PBS), JBNT, FN y las soluciones JBNT/FN NM en un pozo de una diapositiva de 8 pozos, respectivamente.</li>
	<li>Secar el reloj de cubierta de 8 pozos con el instrumento liofilizado para recubrir materiales en la parte inferior de los pozos. El protocolo para el secado por congelación es el mismo que el paso 3.5.</li>
	<li>A continuación, añadir 10.000 células/pozos hMSCs (Pasaje 3) en los pozos e incubarlos durante 24 h a 37 °C con un 5% de CO2.</li>
	<li>Después de la incubación, pipetear el medio de cultivo celular de los pozos y enjuagar el cultivo con solución PBS 1x.</li>
	<li>Añadir 100 μL de la solución fija a cada pozo con células y dejar durante 5 min. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Diluir 1% Tritón-X 100 a 0.1%. Añadir 100 μL de 0.1% Triton-X 100 a cada pozo con células y dejar por 10 min. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Diluir la ródamina fhalloidina a 1,65 μM. Añadir 100 μL de ródamina faloides a cada pozo con células e incubar el reloj de cubierta de cámara manteniendo la luz a temperatura ambiente durante 30 minutos. Enjuague las células suavemente con 1x PBS dos veces.</li>
	<li>Capture las imágenes celulares con un microscopio fluorescente.</li>
	<li>Cuente los números de celda en imágenes fluorescentes para cada grupo. Calcule la densidad de adherencia celular promediando tres áreas aleatorias en cada pozo.</li>
	<li>Presente todos los datos como media ± desviación estándar (SD). Realice análisis estadísticos utilizando una prueba T de una cola, seguida de un análisis de la varianza (ANOVA) con p<0.05, que se considera estadísticamente significativa.</li>
</ol>

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V., Khorsand, B., Geary, S. M., Salem, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3D+Printing+of+scaffolds+for+tissue+regeneration+applications.">3D Printing of scaffolds for tissue regeneration applications.</a> Advanced Healthcare Materials. 4 (12), 1742-1762 (2015).</li><li>Shi, W., 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=Structurally+and+functionally+optimized+silk-fibroin-gelatin+scaffold+using+3D+printing+to+repair+cartilage+injury+in+vitro+and+in+vivo.">Structurally and functionally optimized silk-fibroin-gelatin scaffold using 3D printing to repair cartilage injury in vitro and in vivo.</a> Advanced Material. 29, 1701089 (2017).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Low+inflammatory+activation+by+self-assembling+Rosette+nanotubes+in+human+Calu-3+pulmonary+epithelial+cells.">Low inflammatory activation by self-assembling Rosette nanotubes in human Calu-3 pulmonary epithelial cells.</a> Small. 4 (6), 817-823 (2008).</li><li>Journeay, W. S., Suri, S. S., Moralez, J. G., Fenniri, H., Singh, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rosette+nanotubes+show+low+acute+pulmonary+toxicity+in+vivo.">Rosette nanotubes show low acute pulmonary toxicity in vivo.</a> International Journal of Nanomedicine. 3 (3), 373-383 (2008).</li></ol>

El Dr. Yupeng Chen es cofundador de NanoDe Therapeutics, Inc.

En este estudio, desarrollamos un NM biomimético autoensamblado, que se formó con JBNTs e FN inspirados en el ADN. Al preparar la solución JBNT, el polvo liofilizado JBNT debe disolverse en el agua en lugar de PBS porque PBS causará aglomeración de JBNT, lo que inhibe su montaje. Además, el NM también debe ensamblarse en agua si queremos observar las estructuras nano-fibril del NM, porque la sal en PBS se agrupará con fibras NM, lo que puede reducir en gran medida la resolución de las imágenes.
<p class="jo...

Las células madre mesenquimales humanas (hMSCs) han demostrado el potencial de autorretración y auto-diferenciación a lo largo de diferentes linajes mesenquimales, lo que ayuda en la regeneración y mantenimiento de los tejidos1. Sobre la base del potencial de diferenciación, hMSCs se consideran como candidatos para lesiones de tejido mesenquimal y terapia de trastorno hematopoyético2. hMSCs han demostrado la capacidad de promover la cicatrización de heridas mediante el aumento de la reparación de tejidos, la angiogénesis, y la reducción de la inflamación3. Sin embargo, sin asistencia bioquímica o biomateriales, la eficiencia para que los hMSCs alcancen un tejido objetivo y funcionen en la ubicación deseada es baja4. Aunque se han utilizado varios andamios de ingeniería para atraer hMSCs para adherirse a las lesiones, algunos sitios como la fractura de placas de crecimiento, en medio de un hueso largo, no son fácilmente accesibles por los andamios prefabricados convencionales, que pueden no caber perfectamente en un sitio lesionado de forma irregular.
Aquí, hemos desarrollado un nanomaterial biomimético que puede autoensamblarse in situ y ser inyectado a un área objetivo de difícil acceso. El nm bio-andamio inyectable se compone de nanotubos base Janus (JBNT) y fibronectina (FN). Los JBNT, también conocidos como los Rosette Nanotubes (RNTs), se derivan de pares de bases de ADN, específicamente timina y adenina, aquí5,6,7. Como se ve en la Figura 1,los nanotubos se forman cuando seis moléculas de la base de ADN derivada se autoensamblan a través de enlaces de hidrógeno para formar un plano6. Seis moléculas se apilan unas sobre otras en un plano a través de una fuerte interacción de pi-stacking7,que puede ser de hasta 200-300 μm de longitud. Los JBNT están diseñados para imitar morfológicamente las fibras de colágeno para que el FN reaccione con ellas.
FN es una glicoproteína adhesiva de alto peso molecular, que se puede encontrar en la matriz extracelular (ECM)9. Estos pueden mediar el apego de las células madre a otros componentes del ECM, particularmente el colágeno10. Diseñamos JBNT para imitar morfológicamente las fibras de colágeno para que FN pueda reaccionar con ellas para formar NM en pocos segundos a través de la unión no covalente. Por lo tanto, NM es un prometedor bio-andamio para ser inyectado en un sitio de fractura ósea que no podría ser accesible por los andamios fabricados convencionalmente. Aquí, el NM inyectable presenta una excelente capacidad para mejorar el anclaje hMSC in vitro, exhibiendo su potencial para servir como un andamio para la regeneración de tejidos.

<table>
	<tbody>
		<tr>
			<td>1,2-dichloroethane</td>
			<td>Alfa Aesar</td>
			<td>39121</td>
			<td></td>
		</tr>
		<tr>
			<td>2-cyanoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>C88505</td>
			<td></td>
		</tr>
		<tr>
			<td>4-Dimethylaminopyridine</td>
			<td>TCI America</td>
			<td>D1450</td>
			<td></td>
		</tr>
		<tr>
			<td>8 wells Chambered Coverglass</td>
			<td>Thermo Fisher</td>
			<td>155409</td>
			<td></td>
		</tr>
		<tr>
			<td>96-well plate</td>
			<td>Corning</td>
			<td>353072</td>
			<td></td>
		</tr>
		<tr>
			<td>absolute ethanol</td>
			<td>Thermo Fisher</td>
			<td>BP2818500</td>
			<td></td>
		</tr>
		<tr>
			<td>acetone</td>
			<td>Sigma-Aldrich</td>
			<td>179124</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td>34851</td>
			<td></td>
		</tr>
		<tr>
			<td>allylamine</td>
			<td>Sigma-Aldrich</td>
			<td>145831</td>
			<td></td>
		</tr>
		<tr>
			<td>Basic Plasma Cleaner</td>
			<td>Harrick Plasma</td>
			<td>PDC32G</td>
			<td></td>
		</tr>
		<tr>
			<td>citric acid</td>
			<td>Sigma-Aldrich</td>
			<td>251275</td>
			<td></td>
		</tr>
		<tr>
			<td>concentrated hydrochloric acid</td>
			<td>Sigma-Aldrich</td>
			<td>H1758</td>
			<td></td>
		</tr>
		<tr>
			<td>Deionized water</td>
			<td>Thermo Fisher</td>
			<td>15230147</td>
			<td></td>
		</tr>
		<tr>
			<td>dichloromethane</td>
			<td>Sigma-Aldrich</td>
			<td>270997</td>
			<td></td>
		</tr>
		<tr>
			<td>diethyl ether</td>
			<td>Sigma-Aldrich</td>
			<td>296082</td>
			<td></td>
		</tr>
		<tr>
			<td>Di-tert-butyl dicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>361941</td>
			<td></td>
		</tr>
		<tr>
			<td>ethyl acetate</td>
			<td>Sigma-Aldrich</td>
			<td>319902</td>
			<td></td>
		</tr>
		<tr>
			<td>ethylcarbamate</td>
			<td>Sigma-Aldrich</td>
			<td>U2500</td>
			<td></td>
		</tr>
		<tr>
			<td>Fibronectin</td>
			<td>Thermo Fisher</td>
			<td>PHE0023</td>
			<td></td>
		</tr>
		<tr>
			<td>Fixative Solution (4 % formaldehyde prepared in PBS)</td>
			<td>Thermo Fisher</td>
			<td>R37814</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidinium hydrochloride</td>
			<td>Alfa Aesar</td>
			<td>A13543</td>
			<td></td>
		</tr>
		<tr>
			<td>hexanes</td>
			<td>Sigma-Aldrich</td>
			<td>227064</td>
			<td></td>
		</tr>
		<tr>
			<td>Human mesenchymal stem cells</td>
			<td>Lonza</td>
			<td>PT-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td></td>
		</tr>
		<tr>
			<td>methyl iodide</td>
			<td>Sigma-Aldrich</td>
			<td>289566</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-Diisopropylethylamine</td>
			<td>Alfa Aesar</td>
			<td>A17114</td>
			<td></td>
		</tr>
		<tr>
			<td>N,N-dimethylformamide</td>
			<td>Sigma-Aldrich</td>
			<td>227056</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Methylmorpholine N-oxide</td>
			<td>Alfa Aesar</td>
			<td>A19802</td>
			<td></td>
		</tr>
		<tr>
			<td>Osmium tetraoxide</td>
			<td>Alfa Aesar</td>
			<td>45385</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>Thermo Fisher</td>
			<td>15140163</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate Buffer Solution</td>
			<td>Thermo Fisher</td>
			<td>20012050</td>
			<td></td>
		</tr>
		<tr>
			<td>phosphoryl chloride</td>
			<td>Sigma-Aldrich</td>
			<td>201170</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium carbonate</td>
			<td>Sigma-Aldrich</td>
			<td>347825</td>
			<td></td>
		</tr>
		<tr>
			<td>reverse phase column</td>
			<td>Thermo Fisher</td>
			<td>25305-154630</td>
			<td></td>
		</tr>
		<tr>
			<td>Rhodamine Phalloidin</td>
			<td>Thermo Fisher</td>
			<td>R415</td>
			<td></td>
		</tr>
		<tr>
			<td>silica gel</td>
			<td>TCI America</td>
			<td>S0821</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate</td>
			<td>Sigma-Aldrich</td>
			<td>S6014</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium ethoxide</td>
			<td>Alfa Aesar</td>
			<td>L13083</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium periodide</td>
			<td>Sigma-Aldrich</td>
			<td>71859</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfate</td>
			<td>Sigma-Aldrich</td>
			<td>239313</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium sulfite</td>
			<td>Sigma-Aldrich</td>
			<td>S0505</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium triacetoxyborohydride</td>
			<td>Alfa Aesar</td>
			<td>B22060</td>
			<td></td>
		</tr>
		<tr>
			<td>spectrophotometer(NanoDrop One/One&#7580; UV-Vis)</td>
			<td>Thermo Fisher</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Growth Medium BulletKit</td>
			<td>Lonza</td>
			<td>PT-3001</td>
			<td></td>
		</tr>
		<tr>
			<td>tetrahydrofuran</td>
			<td>Sigma-Aldrich</td>
			<td>401757</td>
			<td></td>
		</tr>
		<tr>
			<td>thioanisole</td>
			<td>Sigma-Aldrich</td>
			<td>T28002</td>
			<td></td>
		</tr>
		<tr>
			<td>toluene</td>
			<td>Sigma-Aldrich</td>
			<td>179418</td>
			<td></td>
		</tr>
		<tr>
			<td>triethylamine</td>
			<td>Alfa Aesar</td>
			<td>A12646</td>
			<td></td>
		</tr>
		<tr>
			<td>trifluoroacetic acid</td>
			<td>Alfa Aesar</td>
			<td>A12198</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Thermo Fisher</td>
			<td>HFH10</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA solution</td>
			<td>Thermo Fisher</td>
			<td>25200056</td>
			<td></td>
		</tr>
	</tbody>
</table>

fabrication of a biomimetic nano-matrix with janus base nanotubes and fibronectin for stem cell adhesion

Un NM biomimético fue desarrollado para servir como un andamio biológico de ingeniería de tejidos, que puede mejorar el anclaje de las células madre. El NM biomimético se forma a partir de JBNT y FN a través del autoensamblaje en una solución acuosa. Los JBNT miden 200-300 μm de longitud con canales huecos hidrofóbicos internos y superficies hidrófilos externas. Los JBNT se cobran positivamente y los FNs se cobran negativamente. Por lo tanto, cuando se inyectan en una solución acuosa neutral, se unen a través de la unión novalente para formar los paquetes NM. El proceso de autoensamblaje se completa en pocos segundos sin ningún iniciador químico, fuente de calor o luz UV. Cuando el pH de la solución NM es menor que el punto isoeléctrico de los FN (pI 5.5-6.0), los paquetes NM se auto-liberarán debido a la presencia de FN cargado positivamente.
NM es conocido por imitar la matriz extracelular (ECM) morfológicamente y por lo tanto, se puede utilizar como un andamio inyectable, que proporciona una excelente plataforma para mejorar la adhesión hMSC. El análisis de densidad celular y los experimentos de imágenes de fluorescencia indicaron que los NMs aumentaron significativamente el anclaje de los hMSC en comparación con el control negativo.

Nuestros estudios descubrieron que la formación del NM de JBNT y FN es rápida, lo que sucedió en 10 segundos. Como se muestra en la Figura 2,floccule blanco se obtuvo cuando la solución JBNT se mezcló con la solución FN y se pipeted varias veces. El proceso de formación de NM es completamente biomimético. No se necesitan estímulos externos. El proceso de fabricación es mucho más fácil que el de algunos biomateriales emergentes, que se basa en la luz ultravioleta o iniciador quím...

Watch this Scientific Journal Video about Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion at JoVE.com

Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

El objetivo de este protocolo es mostrar el montaje de una nanomatrix biomimética (NM) con nanotubos base Janus (JBNT) y fibronectina (FN). Cuando se co-cultivan con células madre mesenquimales humanas (hMSCs), los NMs exhiben una excelente bioactividad en el fomento de la adhesión a los hMSCs.

Fabricación de una nano-matriz biomimética con nanotubos base Janus y fibronectina para adherencia a células madre

A biomimetic NM was developed to serve as a tissue-engineering biological scaffold, which can enhance stem cell anchorage. The biomimetic NM is formed ...

fabrication-biomimetic-nano-matrix-with-janus-base-nanotubes

Research

JoVE Journal

Bioengineering

4.0K vistas.  University of Connecticut. El objetivo de este protocolo es mostrar el montaje de una nanomatrix biomimética (NM) con nanotubos base Janus (JBNT) y fibronectina (FN). Cuando se co-cultivan con células madre mesenquimales humanas (hMSCs), los NMs exhiben una excelente bioactividad en el fomento de la adhesión a los hMSCs.

Video: Fabrication of a Biomimetic Nano-Matrix with Janus Base Nanotubes and Fibronectin for Stem Cell Adhesion

Microfluidic Fabrication of Polymeric and Biohybrid Fibers with Predesigned Size and Shape

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and used to dictate the shape as well as the diameter of a core stream.&#160;Grooves in the top and bottom of a microfluidic channel were designed to direct the sheath fluid and shape the core fluid.&#160;By matching the viscosity and hydrophilicity of the sheath and core fluids, the interfacial effects are minimized and complex fluid shapes can be formed.&#160;Controlling the relative flow rates of the sheath and core fluids determines the cross-sectional area of the core fluid.&#160;Fibers have been produced with sizes ranging from 300 nm to ~1 mm, and fiber cross-sections can be round, flat, square, or complex as in the case with double anchor fibers. Polymerization of the core fluid downstream from the shaping region solidifies the fibers.&#160;Photoinitiated click chemistries are well suited for rapid polymerization of the core fluid by irradiation with ultraviolet light.&#160;Fibers with a wide variety of shapes have been produced from a list of polymers including liquid crystals, poly(methylmethacrylate), thiol-ene and thiol-yne resins, polyethylene glycol, and hydrogel derivatives. Minimal shear during the shaping process and mild polymerization conditions also makes the fabrication process well suited for encapsulation of cells and other biological components.

Two adjacent fluids passing through a grooved microfluidic channel can be directed to form a sheath around a prepolymer core; thereby&#160;determining both shape and cross-section. Photoinitiated polymerization, such as thiol click chemistry, is well suited for rapidly solidifying the core fluid into a microfiber with predetermined size and shape.

Fabricación Microfluídica De Fibras Poliméricas Y Biohíbridas Con Tamaño y Forma Prediseñados

A &#8220;sheath&#8221; fluid passing through a microfluidic channel at low Reynolds number can be directed around another &#8220;core&#8221; stream and ...

Investigación

Bioingeniería

Helical Organization of Blood Coagulation Factor VIII on Lipid Nanotubes

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and membrane environment2.
Coagulation Factor VIII (FVIII)3 is a multi-domain blood plasma glycoprotein. Defect or deficiency of FVIII is the cause for Hemophilia type A&#160;-&#160;a severe bleeding disorder. Upon proteolytic activation, FVIII binds to the serine protease Factor IXa on the negatively charged platelet membrane, which is critical for normal blood clotting4. Despite the pivotal role FVIII plays in coagulation, structural information for its membrane-bound state is incomplete5. Recombinant FVIII concentrate is the most effective drug against Hemophilia type A and commercially available FVIII can be expressed as human or porcine, both forming functional complexes with human Factor IXa6,7.
In this study we present a combination of Cryo-electron microscopy (Cryo-EM), lipid nanotechnology and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.&#160;The representative results demonstrate that our approach is sufficiently sensitive to define the differences in the helical organization between the two highly homologous in sequence (86% sequence identity) proteins. Detailed protocols for the helical organization, Cryo-EM and electron tomography (ET) data acquisition are given. The two-dimensional (2D) and three-dimensional (3D) structure analysis applied to obtain the 3D reconstructions of human and porcine FVIII-LNT is discussed. The presented human and porcine FVIII-LNT structures show the potential of the proposed methodology to calculate the functional, membrane-bound organization of blood coagulation Factor VIII at high resolution.

We present a combination of Cryo-electron microscopy, lipid nanotechnology, and structure analysis applied to resolve the membrane-bound structure of two highly homologous FVIII forms: human and porcine. The methodology developed in our laboratory&#160;to helically organize the two functional recombinant FVIII forms on negatively charged lipid nanotubes (LNT) is described.

Organización helicoidal de sangre Coagulación Factor VIII en lípidos nanotubos

Cryo-electron microscopy (Cryo-EM)1 is a powerful approach to investigate the functional structure of proteins and complexes in a hydrated state and ...

Using Cell-substrate Impedance and Live Cell Imaging to Measure Real-time Changes in Cellular Adhesion and De-adhesion Induced by Matrix Modification

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and other matrix-modifying oxidants/enzymes released during inflammation) are implicated in triggering pathological changes in cellular function, phenotype and viability in a number of diseases. Here, we describe how cell-substrate impedance and live cell imaging approaches can be readily employed to accurately quantify real-time changes in cell adhesion and de-adhesion induced by matrix modification (using endothelial cells and myeloperoxidase as a pathophysiological matrix-modifying stimulus) with high temporal resolution and in a non-invasive manner. The xCELLigence cell-substrate impedance system continuously quantifies the area of cell-matrix adhesion by measuring the electrical impedance at the cell-substrate interface in cells grown on gold microelectrode arrays. Image analysis of time-lapse differential interference contrast movies quantifies changes in the projected area of individual cells over time, representing changes in the area of cell-matrix contact. Both techniques accurately quantify rapid changes to cellular adhesion and de-adhesion processes. Cell-substrate impedance on microelectrode biosensor arrays provides a platform for robust, high-throughput measurements. Live cell imaging analyses provide additional detail regarding the nature and dynamics of the morphological changes quantified by cell-substrate impedance measurements. These complementary approaches provide valuable new insights into how myeloperoxidase-catalyzed oxidative modification of subcellular extracellular matrix components triggers rapid changes in cell adhesion, morphology and signaling in endothelial cells. These approaches are also applicable for studying cellular adhesion dynamics in response to other matrix-modifying stimuli and in related adherent cells (e.g., epithelial cells).

Here, we present a protocol to continuously quantify cell adhesion and de-adhesion processes with high temporal resolution in a non-invasive manner by cell-substrate impedance and live cell imaging analyses. These approaches reveal the dynamics of cell adhesion/de-adhesion processes triggered by matrix modification and their temporal relationship to adhesion-dependent signaling events.

Utilizando Impedancia Cell-sustrato y imágenes de células vivas para medir los cambios en tiempo real en la adhesión celular y De-adhesión inducidas por Matrix Modificación

Cell-matrix adhesion plays a key role in controlling cell morphology and signaling. Stimuli that disrupt cell-matrix adhesion (e.g., myeloperoxidase and ...

Fabrication of a Functionalized Magnetic Bacterial Nanocellulose with Iron Oxide Nanoparticles

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide nanoparticles (IONP) (Fe3O4) to yield a magnetic bacterial nanocellulose (MBNC). The synthesis of MBNC is a precise and specifically designed multi-step process. Briefly, bacterial nanocellulose (BNC) pellicles are formed from preserved G. xylinus strain according to our experimental requirements of size and morphology. A solution of iron(III) chloride hexahydrate (FeCl3&#183;6H2O) and iron(II) chloride tetrahydrate (FeCl2&#183;4H2O) with a 2:1 molar ratio is prepared and diluted in deoxygenated high purity water. A BNC pellicle is then introduced in the vessel with the reactants. This mixture is stirred and heated at 80 &#176;C in a silicon oil bath and ammonium hydroxide (14%) is then added by dropping to precipitate the ferrous ions into the BNC mesh. This last step allows forming in situ magnetite nanoparticles (Fe3O4) inside the bacterial nanocellulose mesh to confer magnetic properties to BNC pellicle. A toxicological assay was used to evaluate the biocompatibility of the BNC-IONP pellicle. Polyethylene glycol (PEG) was used to cover the IONPs in order to improve their biocompatibility. Scanning electron microscopy (SEM) images showed that the IONP were located preferentially in the fibril interlacing spaces of the BNC matrix, but some of them were also found along the BNC ribbons. Magnetic force microscope measurements performed on the MBNC detected the presence magnetic domains with high and weak intensity magnetic field, confirming the magnetic nature of the MBNC pellicle. Young&#39;s modulus values obtained in this work are also in a reasonable agreement with those reported for several blood vessels in previous studies.

Here, we present a protocol to make a bacterial nanocellulose (BNC) magnetic for applications in damaged blood vessel reconstruction. The BNC was synthesized by G. xylinus strain. On the other hand, magnetization of the BNC was realized through in situ precipitation of Fe2+ and Fe3+ ferrous ions inside the BNC mesh.

La fabricación de una funcionalizado magnética bacteriana NANOCELULOSA con nanopartículas de óxido de hierro

In this study, bacterial nanocellulose (BNC) produced by the bacteria Gluconacetobacter xylinus is synthesized and impregnated in situ with iron oxide ...

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer (SALB) Method

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related processes while conferring biocompatibility and biofunctionality to solid substrates. The spontaneous adsorption and rupture of phospholipid vesicles is the most commonly used method to form SLBs. However, under physiological conditions, vesicle fusion (VF) is limited to only a subset of lipid compositions and solid supports. Here, we describe a one-step general procedure called the solvent-assisted lipid bilayer (SALB) formation method in order to form SLBs which does not require vesicles. The SALB method involves the deposition of lipid molecules onto a solid surface in the presence of water-miscible organic solvents (e.g., isopropanol) and subsequent solvent-exchange with aqueous buffer solution in order to trigger SLB formation. The continuous solvent exchange step enables application of the method in a flow-through configuration suitable for monitoring bilayer formation and subsequent alterations using a wide range of surface-sensitive biosensors. The SALB method can be used to fabricate SLBs on a wide range of hydrophilic solid surfaces, including those which are intractable to vesicle fusion. In addition, it enables fabrication of SLBs composed of lipid compositions which cannot be prepared using the vesicle fusion method. Herein, we compare results obtained with the SALB and conventional vesicle fusion methods on two illustrative hydrophilic surfaces, silicon dioxide and gold. To optimize the experimental conditions for preparation of high quality bilayers prepared via the SALB method, the effect of various parameters, including the type of organic solvent in the deposition step, the rate of solvent exchange, and the lipid concentration is discussed along with troubleshooting tips. Formation of supported membranes containing high fractions of cholesterol is also demonstrated with the SALB method, highlighting the technical capabilities of the SALB technique for a wide range of membrane configurations.

Biomembrane Fabrication by the Solvent-assisted Lipid Bilayer SALB Method

We present an experimental protocol to form a supported lipid bilayer on solid substrates without using lipid vesicles. We demonstrate a one-step method to form a lipid bilayer on silicon dioxide and gold as well as supported membranes with cholesterol-enriched domain for various biological applications.

Biomembrana Fabrication por la bicapa lipídica (SALB) Método-Solvente asistida

In order to mimic cell membranes, the supported lipid bilayer (SLB) is an attractive platform which enables in vitro investigation of membrane-related ...

Micropatterning and Assembly of 3D Microvessels

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass based substrates, and hydrogel-based tube formation assays. These assays, while informative, do not recapitulate lumen geometry, proper extracellular matrix, and multi-cellular proximity, which play key roles in modulating vascular function. This manuscript describes an injection molding method to generate engineered vessels with diameters on the order of 100 &#181;m. Microvessels are fabricated by seeding endothelial cells in a microfluidic channel embedded within a native type I collagen hydrogel. By incorporating parenchymal cells within the collagen matrix prior to channel formation, specific tissue microenvironments can be modeled and studied. Additional modulations of hydrodynamic properties and media composition allow for control of complex vascular function within the desired microenvironment. This platform allows for the study of perivascular cell recruitment, blood-endothelium interactions, flow response, and tissue-microvascular interactions. Engineered microvessels offer the ability to isolate the influence from individual components of a vascular niche and precisely control its chemical, mechanical, and biological properties to study vascular biology in both health and disease.

This manuscript presents an injection molding method to engineer microvessels that recapitulate physiological properties of endothelium. The microfluidic-based process creates patent 3D vascular networks with tailorable conditions, such as flow, cellular composition, geometry, and biochemical gradients. The fabrication process and examples of potential applications are described.

Micropatterning y Montaje de Microvasos 3D

In vitro platforms to study endothelial cells and vascular biology are largely limited to 2D endothelial cell culture, flow chambers with polymer or glass ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubos de ADN como una herramienta versátil para el estudio de polímeros Semiflexible

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

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

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

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

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

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

Fabrication of Custom Agarose Wells for Cell Seeding and Tissue Ring Self-assembly Using 3D-Printed Molds

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease modeling. Self-assembled tissues offer advantages over scaffold-based tissue engineering, such as enhanced matrix deposition, strength, and function. However, there are few available methods for fabricating 3D tissues without seeding cells on or within a supporting scaffold. Previously, we developed a system for fabricating self-assembled tissue rings by seeding cells into non-adhesive agarose wells. A polydimethylsiloxane (PDMS) negative was first cast in a machined polycarbonate mold, and then agarose was gelled in the PDMS negative to create ring-shaped cell seeding wells. However, the versatility of this approach was limited by the resolution of the tools available for machining the polycarbonate mold. Here, we demonstrate that 3D-printed plastic can be used as an alternative to machined polycarbonate for fabricating PDMS negatives. The 3D-printed mold and revised mold design is simpler to use, inexpensive to produce, and requires significantly less agarose and PDMS per cell seeding well. We have demonstrated that the resulting agarose wells can be used to create self-assembled tissue rings with customized diameters from a variety of different cell types. Rings can then be used for mechanical, functional, and histological analysis, or for fabricating larger and more complex tubular tissues.

This protocol describes a platform for fabricating self-assembled tissue rings in variable sizes using a customized 3D-printed plastic mold. PDMS negatives are cured in the 3D-printed mold; then agarose is cast in the cured PDMS negatives. Cells are seeded into the resulting agarose wells where they aggregate into tissue rings.

Fabricación de pozos de agarosa personalizado para la siembra de células y tejido anillo autoensamblaje con moldes impresa en 3D

Engineered tissues are being used clinically for tissue repair and replacement, and are being developed as tools for drug screening and human disease ...

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are an emerging treatment for photoreceptor degenerative diseases that seek to restore vision by artificially stimulating the surviving retinal neurons in the hope of eliciting comprehensible visual perception in patients. Current retinal prostheses have demonstrated success in restoring limited vision to patients using an array of electrodes to electrically stimulate the retina but face substantial physical barriers in restoring high acuity, natural vision to patients. Chemical neurostimulation using native neurotransmitters is a biomimetic alternative to electrical stimulation and could bypass the fundamental limitations associated with retinal prostheses using electrical neurostimulation. Specifically, chemical neurostimulation has the potential to restore more natural vision with comparable or better visual acuities to patients by injecting very small quantities of neurotransmitters, the same natural agents of communication used by retinal chemical synapses, at much finer resolution than current electrical prostheses. However, as a relatively unexplored stimulation paradigm, there is no established protocol for achieving chemical stimulation of the retina in vitro. The purpose of this work is to provide a detailed framework for accomplishing chemical stimulation of the retina for investigators who wish to study the potential of chemical neuromodulation of the retina or similar neural tissues in vitro. In this work, we describe the experimental setup and methodology for eliciting retinal ganglion cell (RGC) spike responses similar to visual light responses in wild-type and photoreceptor-degenerated wholemount rat retinas by injecting controlled volumes of the neurotransmitter glutamate into the subretinal space using glass micropipettes and a custom multiport microfluidic device. This methodology and protocol are general enough to be adapted for neuromodulation using other neurotransmitters or even other neural tissues.

Methodology for Biomimetic Chemical Neuromodulation of Rat Retinas with the Neurotransmitter Glutamate In Vitro

This protocol describes a novel method for investigating a form of chemical neurostimulation of wholemount rat retinas in vitro with the neurotransmitter glutamate. Chemical neurostimulation is a promising alternative to the conventional electrical neurostimulation of retinal neurons for treating irreversible blindness caused by photoreceptor degenerative diseases.

Metodología para la neuromodulación química Biomimetic de Retinas de ratas con el neurotransmisor glutamato In Vitro

Photoreceptor degenerative diseases cause irreparable blindness through the progressive loss of photoreceptor cells in the retina. Retinal prostheses are ...