The authors thank the Fraunhofer ISC for the collaboration concerning the construction of the automated wounding device. The project was founded by Fraunhofer internal project &#8220;M&#228;rkte von &#220;bermorgen&#8221; (SkinHeal).

NOTA: El protocolo está diseñado para la producción de 24 equivalentes de piel de espesor completo. Fibroblastos dérmicos y queratinocitos epidérmicos humanos fueron aisladas de biopsias de piel de acuerdo con un protocolo previamente publicado. 21,22 consentimiento informado se obtuvo de antemano y el estudio fue aprobado por el comité de ética institucional en la investigación humana de las Julius-Maximilians-Universität Würzburg (voto 182 / 10). 1. Producción de Dérmica Componente <ol><li> Disolver colágeno con ácido acético al 0,1% a una concentración final de 6 mg / ml. Para la solución de neutralización gel, mezclar 232,5 ml 2x DMEM, 7,5 ml de suero de ternera fetal, 7,5 ml 3 M HEPES, 2,5 ml de sulfato de condroitina (5 mg / ml). Mantenga solución de neutralización de gel y solución de colágeno en el hielo. Calcule 500 l de gel por equivalente de inserción / piel. NOTA: A medida que la solución de neutralización gel se mezclará con dos partes de solución de colágeno prepare doble cantidad de colágeno. </li><li> Coloque 24 inserciones en una placa de 24 pocillos. </li><li> Resuspender los fibroblastos dérmicos humanos (HDF) en 10 ml de DMEM, centrifugar durante 5 min a 270 xg y contar las células. Extraer la cantidad necesaria de HDF (5 x 10 4 células por 500 gel de colágeno l) y centrifugar las células durante 5 min a 270 x g. </li><li> Eliminar el sobrenadante y resuspender el cuidado HDF en 4 ml enfriados solución de neutralización gel sin producir burbujas de aire. Desde este paso asegúrese de trabajar tan rápido como sea posible para evitar la solidificación prematura de los geles de colágeno. </li><li> Resuspender la solución con 8 ml de solución de colágeno. </li><li> Tome el colágeno de células-mezcla con múltiples pasos-pipeta y llenar rápidamente 500 l de mezcla en cada inserción. Incubar los geles durante 20 min en una incubadora (37 ° C, 5% de CO 2) para cuajar los geles. </li><li> Sumergir los geles en ml Medio Eagle Modificado de Dulbecco (DMEM) + suero de 2 10% de ternera fetal (FCS) por pocilloy se incuba durante 24 horas en una incubadora (37 ° C, 5% de CO 2). </li></ol> 2. La adición de queratinocitos epidérmicos humanos (HEK) <ol><li> Retire medio después de 24 horas. Disolver proteína humana fibronectina con agua ultrapura hasta una concentración final de 50 mg / ml. Cubrir cada equivalente dérmico con 25 l de solución de fibronectina. Incubar los geles durante 30 minutos en una incubadora (37 ° C, 5% de CO 2). </li><li> Mientras tanto, resuspender el HEK en 10 ml KGM-2 y establecer la concentración de células a 1 x 10 6 células / ml con KGM-2 listo + 5% de FCS. Semilla de 1 x 10 5 células HEK en cada gel. Incubar los geles durante 45 minutos en la incubadora (37 ° C, 5% de CO 2) para permitir que las células se adhieran. </li><li> Sumergir los geles con KGM-2 listo + 5% de FCS y la cultura en la incubadora (37 ° C, 5% de CO 2). </li></ol> 3. Cultura de equivalentes de piel de grosor completo (FTSE) <ol><li> FTSE Cultura durante 6-7 díasdescendente en FCS-concentración (5%, 2%, y 0% de FCS). Cambie el medio cada 2-3 días con la siguiente concentración más baja de FCS. Para ello, eliminar completamente el medio y añadir 1,6 ml de medio fresco. </li><li> El día 7, eliminar por completo medio. NOTA: No toque la superficie FTSE con la punta de la pipeta mientras lo hace. </li><li> Coloque todos los inserte en 1 pocillo de una placa de 6 pocillos con pinzas estériles. Añadir 1,5 ml de medio de transporte aéreo por pozo, asegúrese de no mojar la superficie FTSE. Cambie medio cada 2-3 días para otros 14 días. NOTA: El nivel de llenado del medio es hasta el menisco de la FTSE. </li></ol> 4. Análisis histológico <ol><li> Fijación y de inclusión en parafina de la FTSE <ol><li> Retire medio de cultivo, transferir los insertos en placas de 24 pocillos frescos y fijar los tejidos mediante la adición de 1,6 ml de paraformaldehído al 4% a cada pocillo e incubar los tejidos durante 2 horas a RT. ¡Precaución! Paraformaldehyd es tóxico, manipular bajo campana de humos. TransferenciaEl FTSE a un cassette de la incrustación de tejidos. Retirar el resto de fijador por lavado con agua y deshidratar el tejido utilizando concentraciones de etanol ascendente. </li><li> Divida el equivalente de piel por un corte vertical en el centro. Coloque las dos piezas en un molde base de metal parafina lleno de superficies de corte hacia abajo. Añadir el casete de tejido en la parte superior del molde como un respaldo. </li><li> Cortar 3-5 micras rodajas y flotar en un baño de agua a 40 ° C para straitening, montar toboganes en portaobjetos de microscopio adecuados. Rebanadas seque completamente. </li></ol></li><li> Hematoxilina y eosina (H &amp; E) y de inmunohistoquímica (IHC) tinción <ol><li> Lugar desliza en estante y se incuba durante 1 hora a 60 ° C. Asegúrese de que la parafina se funde. Manchas rebanadas con H &amp; E rehidrata como tinción resumen estandarizado. </li><li> Para la tinción de H &amp; E preparar 4x cubetas de tinción de vidrio con xilol, 3x con etanol 96%, 2 veces con etanol al 70%, 1x con etanol al 50%, 4x agua desionizada, hematoxilina 1x, 1x HCl-eTHANOL (13,7 ml de HCl 1 M ad 200 ml de etanol 50%), 1x agua de la lengüeta, 1x eosina (1 g de eosina en 100 ml de agua desionizada) y 2x 2-propanol. </li><li> Lugar desliza 10 min en xilol I y 10 min en xilol II Desparafinar y rehidratar diapositivas. Dip desliza 3x en etanol 96% I, 3x en etanol 96% II, 3x en etanol al 70% y 3x en etanol al 50%. Gire las diapositivas de agua desionizada. Para diferenciar colorante hematoxilina, 2x baño en HCl-etanol. Enjuague con agua desionizada. Para azulado, lugar desliza 5 minutos en agua ficha. Para la tinción de eosina, lugar desliza 1 min en eosina y lavar después en agua destilada. Para la deshidratación, los portaobjetos de inmersión 2x ​​en etanol al 70%, 2x en etanol 96% y las colocan durante 5 min en 2-propanol I, durante 5 min en 2-propanol II, 5 min en xilol I y 5 min en xilol II. Después de la tinción H &amp; E, los núcleos celulares se tiñen de azul, el citoplasma y la matriz extracelular se tiñen de rojo. </li><li> Para la recuperación de antígenos, retire la parafina con xileno y rehidratar rodajas para la tinción. Coloque las rebanadasen la olla de vapor y cocinar rebanadas desparafinados y rehidratados durante 20 min en pre-calentadas 10 mM tampón de citrato (pH 6). </li><li> Coloque los portaobjetos en tampón de lavado (PBS tampón + 0,5%) y polisorbato 20 rebanadas de círculo con un tobogán repelente rotulador líquido o aguja de diamante. </li><li> Coloque las diapositivas en una cámara de humedad. Bloquear la peroxidasa endógena, mediante la adición de 100 l de solución de peróxido de hidrógeno al 3% en cada rebanada. ¡Precaución! Muy peligroso en caso de la piel y el contacto visual. Manejar con equipo de protección personal. Lavar los portaobjetos en tampón de lavado. </li><li> Aplicar anticuerpo primario y se incuba durante 1 hora a RT. Lavar los portaobjetos tres veces con tampón de lavado. A partir de aquí, las muestras se deben mantener en la oscuridad. </li><li> Use sistema de detección de biotina-estreptavidina para la detección de unión a antígeno del anticuerpo específico. </li><li> Contratinción núcleos con hematoxilina durante 1 minuto. Deshidratar y montar toboganes. </li><li> Muestras de imágenes utilizando un microscopio inverso. </li></ol></li></ol> &lt;p class = &quot;jove_title&quot;&gt; 5. Lesiones por el uso del dispositivo automatizado Heridas (AWD) <ol><li> Prepare el AWD desinfectando la zona de trabajo con etanol al 70% y la luz ultravioleta. Asegúrese de que el cabezal de perforación del tamaño deseado (por ejemplo, 1 mm) se adjunta. </li><li> Coloque equivalentes de piel en áreas designadas de la placa de soporte de muestras en autoclave usando pinzas estériles. Coloque la placa de soporte de muestras en la toma de debajo de la cabeza de perforación. </li><li> Utilice el software de control para establecer los parámetros hiriendo la siguiente manera: la velocidad de giro: 15000 rpm; penetración: 1,5 mm; propulsión: 100 Hz. Estos parámetros se deben definir para cada tipo de muestra individual. Transferencia hiriendo parámetros a la tracción total. </li><li> Iniciar procedimiento hiriendo. Retire la placa de soporte de muestras de la toma. Transferencia herido FTSE nuevo en insertos utilizados para el cultivo utilizando pinzas estériles. </li><li> Proceda con la cultura de FTSE heridos en la incubadora (37 ° C, 5% CO 2) para la investigación de la regeneración. Alternatively, utilizar los modelos para el análisis histológico en cualquier momento. </li></ol>

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The authors have nothing to disclose.

En las células in vitro generalmente se expanden en cultivos celulares bidimensionales, en el que las células se adhieren a las superficies de plástico. Sin embargo, estas condiciones de cultivo no reflejan las condiciones en tres dimensiones fisiológicas en las que las células crecen in vivo. En condiciones tridimensionales, las células pueden formar adjuntos célula-célula y célula-matriz naturales y migrar en tres dimensiones. Especialmente en la herida cutánea sanar el parecido de...

La piel es el órgano más grande del cuerpo. Se crea una barrera entre el entorno exterior y los órganos internos. Por otra parte, la piel protege el cuerpo de la pérdida de líquidos, las influencias ambientales, las lesiones y las infecciones y ayuda a regular la temperatura corporal 1. Debido a su ubicación a la vista, la piel es a menudo afectada por los traumatismos mecánicos, térmicos o químicos. Aunque la piel es generalmente capaz de auto-reparación, múltiples factores locales como la infección, la oxigenación y la suficiencia venosa pueden conducir a problemas de cicatrización de la herida. La cicatrización de heridas también puede ser interferido por factores sistémicos como la obesidad, alcoholismo, tabaquismo, medicación, nutrición y las enfermedades tales como la diabetes. 2 El proceso de curación de las heridas puede dividirse en 3 fases: (i) la fase inflamatoria, (ii) la proliferación y la (iii) fase de remodelación. Tras la lesión en la piel, un complejo de señal en cascada se inicia, lo que conduce al cierre de la herida. 3Después de la lesión, la herida está sangrando y se forma un coágulo de sangre. Los fibroblastos se mueven en la coagulación de la sangre y lo reemplazan con nuevo tejido que está remodelado posteriormente durante años. La comprensión actual de la reparación cutánea procesos biológicos subyacentes es limitado. Modelos pequeños animales y de cerdo se han utilizado para estudiar la cicatrización de heridas. Sin embargo, estos resultados no pueden ser transferidos directamente a los seres humanos debido a las diferencias específicas de las especies. Además de estos modelos in vivo, algunos aspectos de la cicatrización de heridas se pueden estudiar mediante la simulación de una situación de la herida a través de rascado de cultivos en monocapa in vitro basado en líneas celulares inmortalizadas o células primarias. 4 Estos modelos rascarse están altamente estandarizados, pero no suficientemente reflejan la . compleja fisiología in vivo 5 Además de los modelos de dos dimensiones, los equivalentes de piel humana en tres dimensiones se han desarrollado para la investigación dermatológica. La parte dérmica de estos modelos son generated utilizando diversos andamios incluyendo dermis descelularizados, 6 hidrogeles de colágeno, glicosaminoglicanos 7,8 9 o materiales sintéticos. 10 Empleando estos equivalentes de piel, el papel de las interacciones epitelio mesenquimal 11, la re-epitelización, la diafonía celular entre fibroblastos y queratinocitos y la influencia de diferentes factores de crecimiento puede ser estudiado. Además, estos modelos son útiles para obtener nuevos conocimientos sobre cómo los fibroblastos migran a la zona herida y cómo los factores quimiotácticos influyen en la regeneración de tejidos. 12 No sólo la generación del modelo de cicatrización de la herida en sí es difícil, sino también para establecer una herida altamente estandarizada en un modelo es problemática. Las técnicas comunes para crear heridas son ensayos de rayado, 13 quemaduras, 14 cintas de abrasión, 15 lesiones térmicas, 16 ampollas de succión, nitrógeno líquido 17, 18, ​​19 lasers </sup&gt; escalpelos, 18 malladores 6 y punzones de biopsia. 20 La mayoría de estos métodos tienen las mismas trampas. Lesiones aplicadas manualmente son difíciles de estandarizar y de reproducirse entre múltiples pruebas. Tamaño, forma y profundidad de la herida varían entre los estudios y por lo tanto perjudican la calidad de los datos de investigación. El uso de láser para heridas de la piel definido puede ser estandarizada con relativa facilidad, pero conduce a una situación imitando heridas de quemaduras. El calor aplicado por láser puede causar desnaturalización de la proteína, la agregación plaquetaria o los vasos constricción, que puede conducir a tejido necrótico. En un enfoque alternativo, hemos desarrollado un dispositivo heridas automatizado (AWD), que nos permite generar heridas cutáneas definidas y precisas en condiciones estériles. Parámetros herida, como la profundidad y la velocidad de penetración, así como las revoluciones de la cabeza de perforación pueden ser reguladas. En este estudio, hemos combinado el AWD con equivalentes de piel de espesor completo en casa desarrollados (ftSE) que son comparables a un protocolo publicado por Gangatirkar et al. 8 La capa dérmica de la piel equivalente se compone de fibroblastos dérmicos humanos (HDF), que están incrustados en un colágeno I hidrogel. En la capa dérmica, los queratinocitos epidérmicos humanos (HEK) se siembran. Dentro de dos semanas en la interfase aire-líquido del HEK construir una epidermis compuesta de varias capas de células vitales y un estrato córneo. Además de la generación de este modelo, este estudio muestra el uso de la tracción total para crear heridas definidos y precisos en FTSE.

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			<td height="21" style="height:21px;width:329px;">Name of Material/ Equipment</td>
			<td style="width:135px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
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			<td></td>
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			<td height="21" style="height:21px;width:329px;">Trypsin EDTA (1:250) 0.5 % in DPBS&#160;</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">L11-003</td>
			<td align="right">0.05%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Dulbecco&#8217;s Phosphate Buffered Saline</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Collagen (6 mg/ml in 0,1 % acetic acid)</td>
			<td style="width:135px;">Produced in house</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibronectin Human Protein, Plasma (50 &#181;g/ml)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">33016-015</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Inserts</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140627</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">6 well plate&#160;</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">140685</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">24 well plate</td>
			<td style="width:135px;">Nunc</td>
			<td align="right" style="width:119px;">142485</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Microscope slides</td>
			<td style="width:135px;">R. Langenbrinck</td>
			<td style="width:119px;">03-0070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fibroblasts culturing (500 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">DMEM, high glucose</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">11965-092</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>10% (50 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinozyten culture medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td>89% (445 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Gel neutralization solution (250 ml):</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Dulbecco&#8217;s Modified Eagle Medium, high Glucose Powder with L-Glutamine</td>
			<td style="width:135px;">PAA</td>
			<td style="width:119px;">G0001,3010</td>
			<td>93% (232,5 ml)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Chondroitin sulfate sodium salt from shark cartilage</td>
			<td style="width:135px;">Sigma</td>
			<td style="width:119px;">C4384-1g</td>
			<td>1% (2,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal bovine serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HEPES</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">H3375-1kg</td>
			<td>3% (7,5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model submers medium (500ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-20111</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2 SupplementPack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Fetal calf serum</td>
			<td style="width:135px;">Bio &#38; Sell</td>
			<td style="width:119px;">FCS.ADD.0500</td>
			<td>5%-2% (25 ml-10 ml)&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Skin model air-liquid interface medium (500 ml):&#160;</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Keratinocyte Growth Medium 2</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:329px;">Keratinocyte Growth Medium 2 Supplement Pack</td>
			<td style="width:135px;">Promocell&#160;</td>
			<td style="width:119px;">C-39011</td>
			<td>adding only supplements: Insulin, Hydrocortisone, Epinephrine, Transferrin, CaCl2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Penicillin-Streptomycin (10,000 U/mL)</td>
			<td style="width:135px;">Life Technologies</td>
			<td style="width:119px;">15140-122</td>
			<td>1% (5 ml)</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:329px;">CaCl2 (300 mM)</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td style="width:119px;">C7902-500g</td>
			<td>&#160;0,62% (3,1 ml)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Histology:</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">IHC-Kit DCS SuperVision 2 HRP</td>
			<td style="width:135px;">DCS</td>
			<td style="width:119px;">PD000KIT</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Vimentin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab92547</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK14 antibody</td>
			<td style="width:135px;">Sigma&#160;</td>
			<td>HPA023040-100&#181;l</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">CK10 antibody</td>
			<td style="width:135px;">Dako</td>
			<td style="width:119px;">M7002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Filaggrin antibody</td>
			<td style="width:135px;">Abcam</td>
			<td>ab81468</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;"></td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">H&#38;E staining</td>
			<td style="width:135px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mayer&#180;s Haemalaun</td>
			<td style="width:135px;">AppliChem</td>
			<td style="width:119px;">A0884,2500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Xylol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>296325-4X2L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Ethanol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>32205-4X2.5L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">HCl</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>H1758-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Eosin</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>E4009-5G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">2-Propanolol</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>I9516-500ML</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:329px;">Mounting Medium</td>
			<td style="width:135px;">Sigma Aldrich</td>
			<td>M1289-10ML</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

El HDF y HEK aislado difieren tanto en la morfología y en la expresión de marcadores típicos. El HDF mostró morfología típica de husillo-forma, mientras que la morfología de células HEK puede ser descrita por una morfología de adoquines. Las células se caracterizan por tinción inmunohistoquímica (Figura 1) antes de utilizarlas por FTSE. El HDF son positivas para vimentina (Figura 1A), un marcador de los fibroblastos. HEK primaria expresar la proteína altamente temprana dife...

Watch this Scientific Journal Video about Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding at JoVE.com

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

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 ...

generation-three-dimensional-full-thickness-skin-equivalent-automated

Research

JoVE Journal

Bioengineering

18.4K vistas.  University Hospital Würzburg. 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. 

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

The Three-Dimensional Human Skin Reconstruct Model: a Tool to Study Normal  Skin and Melanoma Progression

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells behave differently when they are grown within a 3D extra-cellular matrix and also interact with other cells (1-5). Mouse models have been broadly utilized to study tissue morphogenesis in vivo. However mouse and human skin have significant differences in cellular architecture and physiology, which makes it difficult to extrapolate mouse studies to humans. Since melanocytes in mouse skin are mostly localized in hair follicles, they have distinct biological properties from those of humans, which locate primarily at the basal layer of the epidermis. The recent development of 3D human skin reconstruct models has enabled the field to investigate cell-matrix and cell-cell interactions between different cell types. The reconstructs consist of a "dermis" with fibroblasts embedded in a collagen I matrix, an "epidermis", which is comprised of stratified, differentiated keratinocytes and a functional basement membrane, which separates epidermis from dermis. Collagen provides scaffolding, nutrient delivery, and potential for cell-to-cell interaction. The 3D skin models incorporating melanocytic cells recapitulate natural features of melanocyte homeostasis and melanoma progression in human skin. As in vivo, melanocytes in reconstructed skin are localized at the basement membrane interspersed with basal layer keratinocytes. Melanoma cells exhibit the same characteristics reflecting the original tumor stage (RGP, VGP and metastatic melanoma cells) in vivo. Recently, dermal stem cells have been identified in the human dermis (6). These multi-potent stem cells can migrate to the epidermis and differentiate to melanocytes.

In this report, we describe the three-dimensional skin reconstruct model which mimics human skin in architecture and composition. Melanocyte physiology, melanoma progression and the fate of dermal stem cells have been investigated using the skin reconstruct model. The model is also useful as a preclinical tool for drug assessment.

La piel humana tridimensional Reconstrucción del modelo: una herramienta para estudiar la piel normal y la progresión del melanoma

Most in vitro studies in experimental skin biology have been done in 2-dimensional (2D) monocultures, while accumulating evidence suggests that cells ...

Investigación

Bioingeniería

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Impresión plana y tridimensional de tintas conductoras

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Investigating the Three-dimensional Flow Separation Induced by a Model Vocal Fold Polyp

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. Polyps and nodules, which are geometric abnormalities that form on the medial surface of the vocal folds, can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Our laboratory has reported particle image velocimetry (PIV) measurements, within an investigation of a model polyp located on the medial surface of an in&#160;vitro driven vocal fold model, which show that such a geometric abnormality considerably disrupts the glottal jet behavior. This flow field adjustment is a likely reason for the severe degradation of the vocal quality in patients with polyps. A more complete understanding of the formation and propagation of vortical structures from a geometric protuberance, such as a vocal fold polyp, and the resulting influence on the aerodynamic loadings that drive the vocal fold dynamics, is necessary for advancing the treatment of this pathological condition. The present investigation concerns the three-dimensional flow separation induced by a wall-mounted prolate hemispheroid with a 2:1 aspect ratio in cross flow, i.e. a model vocal fold polyp, using an oil-film visualization technique. Unsteady, three-dimensional flow separation and its impact of the wall pressure loading are examined using skin friction line visualization and wall pressure measurements.

Vocal fold polyps can disrupt vocal fold dynamics and thus can have devastating consequences on a patient&#39;s ability to communicate. Three-dimensional flow separation induced by a wall-mounted model polyp and its impact on the wall pressure loading are examined using particle image velocimetry, skin friction line visualization, and wall pressure measurements.

Investigación De La Separación De Flujo Tridimensional Inducida Por Un Pólipo Modelo De Pliegue Vocal

The fluid-structure energy exchange process for normal speech has been studied extensively, but it is not well understood for pathological conditions. ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Una piel llena de defectos modelo para evaluar la vascularización de Biomateriales<em&gt; En Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

Analysis of Cell Migration within a Three-dimensional Collagen Matrix

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic development, wound healing, and immune responses. However, cell migration is also a key mechanism in cancer enabling these cancer cells to detach from the primary tumor to start metastatic spreading. Within the past years various cell migration assays have been developed to analyze the migratory behavior of different cell types. Because the locomotory behavior of cells markedly differs between a two-dimensional (2D) and three-dimensional (3D) environment it can be assumed that the analysis of the migration of cells that are embedded within a 3D environment would yield in more significant cell migration data. The advantage of the described 3D collagen matrix migration assay is that cells are embedded within a physiological 3D network of collagen fibers representing the major component of the extracellular matrix. Due to time-lapse video microscopy real cell migration is measured allowing the determination of several migration parameters as well as their alterations in response to pro-migratory factors or inhibitors. Various cell types could be analyzed using this technique, including lymphocytes/leukocytes, stem cells, and tumor cells. Likewise, also cell clusters or spheroids could be embedded within the collagen matrix concomitant with analysis of the emigration of single cells from the cell cluster/ spheroid into the collagen lattice. We conclude that the 3D collagen matrix migration assay is a versatile method to analyze the migration of cells within a physiological-like 3D environment.

Cell migration is a biological phenomenon that is involved in a plethora of physiological, such as wound healing and immune responses, and pathophysiological processes, like cancer. The 3D-collagen matrix migration assay is a versatile tool to analyze the migratory properties of different cell types within in a 3D physiological-like environment.

Análisis de la migración de células dentro de una matriz de colágeno tridimensional

The ability to migrate is a hallmark of various cell types and plays a crucial role in several physiological processes, including&#160;embryonic ...

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, ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Tridimensional de la impresión de materiales termoplásticos para crear automatizado de bombas de la jeringuilla con retroalimentación de Control para aplicaciones de microfluídica

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Cultivating a Three-dimensional Reconstructed Human Epidermis at a Large Scale

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process and the characterization of the model is described. Neonatal primary keratinocytes are grown submerged on permeable polycarbonate inserts and lifted to the air-liquid interface three days after seeding. After fourteen days of stimulation with defined growth factors and ascorbic acid in high calcium culture medium, the model is fully differentiated. Histological analysis revealed a completely stratified epidermis, mimicking the morphology of native human skin. To characterize the model and its barrier functions, protein levels and localization specific for early-stage keratinocyte differentiation (i.e., keratin 10), late-stage differentiation (i.e., involucrin, loricrin, and filaggrin) and tissue adhesion (i.e., desmoglein 1), were assessed by immunofluorescence. The tissue barrier integrity was further evaluated by measuring transepithelial electrical resistance. Reconstructed human epidermis was responsive to proinflammatory stimuli (i.e., lipopolysaccharide and tumor necrosis factor alpha), leading to increased cytokine release (i.e., interleukin 1 alpha and interleukin 8). This protocol represents a straightforward and reproducible in vitro method to cultivate reconstructed human epidermis as a tool to assess environmental effects and a broad range of skin-related studies.

This protocol describes a straightforward method to cultivate three-dimensional reconstituted human epidermis in a reproducible and robust manner. Additionally, it characterizes the structure-function relationship of the epidermal barrier model. The biological responses of the reconstituted human epidermis upon proinflammatory stimuli are also presented.

El cultivo de una epidermis humana reconstruida tridimensional a gran escala

A three-dimensional human epidermis model reconstructed from neonatal primary keratinocytes is presented. Herein, a protocol for the cultivation process ...

Generation of a Simplified Three-Dimensional Skin-on-a-chip Model in a Micromachined Microfluidic Platform

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of concept, a simplified and undifferentiated human skin containing a dermal (stromal) and an epidermal (epithelial) compartment has been modelled. To accomplish this, a versatile and robust, vinyl-based device divided into two chambers has been developed, overcoming some of the drawbacks present in microfluidic devices based on polydimethylsiloxane (PDMS) for biomedical applications, such as the use of expensive and specialized equipment or the absorption of small, hydrophobic molecules and proteins. Moreover, a new method based on parallel flow was developed, enabling the in situ deposition of both the dermal and epidermal compartments. The skin construct consists of a fibrin matrix containing human primary fibroblasts and a monolayer of immortalized keratinocytes seeded on top, which is subsequently maintained under dynamic culture conditions. This new microfluidic platform opens the possibility to model human skin diseases and extrapolate the method to generate other complex tissues.

Here, we present a protocol to generate a three-dimensional simplified and undifferentiated skin model using a micromachined microfluidic platform. A parallel flow approach allows the in situ deposition of a dermal compartment for the seeding of epithelial cells on top, all controlled by syringe pumps.

Generación de un modelo tridimensional simplificado de piel en un chip en una plataforma microfluídica micromecanizado

This work presents a new, cost-effective, and reliable microfluidic platform with the potential to generate complex multilayered tissues. As a proof of ...

Generation of Multicue Cellular Microenvironments by UV-Photopatterning of Three-Dimensional Cell Culture Substrates

The extracellular matrix is an important regulator of cell function. Environmental cues existing in the cellular microenvironment, such as ligand distribution and tissue geometry, have been increasingly shown to play critical roles in governing cell phenotype and behavior. However, these environmental cues and their effects on cells are often studied separately using in vitro platforms that isolate individual cues, a strategy that heavily oversimplifies the complex in vivo situation of multiple cues. Engineering approaches can be particularly useful to bridge this gap, by developing experimental setups that capture the complexity of the in vivo microenvironment, yet retain the degree of precision and manipulability of in vitro systems.
This study highlights an approach combining ultraviolet (UV)-based protein patterning and lithography-based substrate microfabrication, which together enable high-throughput investigation into cell behaviors in multicue environments. By means of maskless UV-photopatterning, it is possible to create complex, adhesive protein distributions on three-dimensional (3D) cell culture substrates on chips that contain a variety of well-defined geometrical cues. The proposed technique can be employed for culture substrates made from different polymeric materials and combined with adhesive patterned areas of a broad range of proteins. With this approach, single cells, as well as monolayers, can be subjected to combinations of geometrical cues and contact guidance cues presented by the patterned substrates. Systematic research using combinations of chip materials, protein patterns, and cell types can thus provide fundamental insights into cellular responses to multicue environments.

Traditionally, cell culture is performed on planar substrates that poorly mimic the natural environment of cells in vivo. Here we describe a method to produce cell culture substrates with physiologically relevant curved geometries and micropatterned extracellular proteins, allowing systematic investigations into cellular sensing of these extracellular cues.