The authors acknowledge financial support from the Victorian Government&#8217;s Operational Infrastructure Support Program.

NOTA: placentario debe recogerse de embarazos saludables simples, con una preferencia por las cesáreas electivas plazo. Escrito, el consentimiento informado debe darse para la recogida de su placenta. Su comité de ética de investigación en humanos pertinentes deberían homologación toda la colección y el uso de tejidos humanos. 1. Aislamiento de células epiteliales Amnion <ol style="margin-left: 40px;"><li> Coloque la placenta sobre una superficie estéril dentro de una cabina de seguridad biológica de clase II. </li><li> Uso de madejas estériles solución salina equilibrada (HBSS), lavar tanta sangre como sea posible de la superficie de placenta y membranas. </li><li> Con la placenta colocado con el cordón umbilical hacia arriba, separar mecánicamente el borde exterior de las membranas de amnios y el corion. </li><li> Una vez separados, la tira manualmente la membrana amnios del corion la membrana de la placenta, trabajando alrededor del borde de la membrana, hacia el cordón umbilical. NOTA: Sea CAREFul para eliminar las piezas de la contaminación de la membrana del corion del amnios. </li><li> Usando tijeras estériles, cortar la membrana amnios aproximadamente 2 cm desde la base del cordón umbilical y el lugar en unos 500 ml sellado recipiente que contiene 250 ml de HBSS. </li><li> Lávese bien agitando el frasco sellado que contiene la membrana amnios. </li><li> Retire la membrana amnios del recipiente de recogida utilizando pinzas estériles, y el lugar en una placa de Petri de 15 cm. </li><li> Cortar la membrana amnios en trozos de aproximadamente 5 cm de largo (cuando se mantiene en posición vertical con fórceps), desechando pedazos sangrientos o rasgados. </li><li> Colocar en un contenedor sellado de 500 ml y lavar con 250 ml de HBSS aproximadamente 3-5 veces o hasta que la membrana amnios vuelve translúcida sin sangre contaminante. NOTA: la presencia de sangre puede reducir la eficiencia de la digestión enzimática solución. </li><li> Transfiera todas las piezas de la membrana amnios en un recipiente que contiene 50 ml de la solución de digestión enzimática, teniendo cuidado deminimizar el arrastre de HBSS. </li><li> Incubar las piezas amnios en enzimática digieren solución en un baño de agua o habitación caliente a 37 ° C durante 15 minutos con agitación suave para eliminar la sangre restante. </li><li> Retire las piezas de membrana amnios de la digestión enzimática solución y colocar en un nuevo recipiente estéril. </li><li> Añadir 100 ml de solución enzimática fresco digieren a la membrana amnios y se incuban a 37 ° C durante 60 minutos en un baño de agua o habitación caliente con agitación suave (primera digestión). </li><li> Retire las piezas de membrana amnios y colocar en nueva toma cuidado envase para permitir la digestión enzimática solución para drenar de cada pieza de membrana. NOTA: Esto puede conseguirse arrastrando piezas amnios lo largo del borde interior del recipiente. </li><li> Añadir 100 ml de solución enzimática fresco digieren a las piezas de membranas amnióticas e incubar 60 minutos en un baño de agua o habitación caliente a 37 ° C con agitación suave (segunda digestión). </li><li> Pase el Suspensiòn celulardesde 1,14 Paso a través de un filtro de 70 micras y se centrifuga a 1000 xg durante 10 min. </li><li> Retire y deseche el sobrenadante y resuspender el botón celular en 4 ml de HBSS que contiene 1 mg / ml de inhibidor a base de soja de la enzima. Pipetear suavemente varias veces con una pipeta de 1 ml para obtener una suspensión de células individuales. NOTA: En esta etapa las células se pueden dejar en hielo hasta que las células de la segunda digestión se encuentran en la misma etapa de procesamiento. </li><li> Retire las piezas de membrana amnios y colocar en nueva toma cuidado envase para permitir la digestión enzimática solución para drenar de cada pieza de membrana. NOTA: Esto puede conseguirse arrastrando piezas amnios lo largo del borde interior del recipiente. </li><li> Pasar la suspensión de células a partir del paso a través de un filtro de 1,18 micras y 70 centrifugar a 1000 xg durante 10 min. </li><li> Retire y deseche el sobrenadante y resuspender el botón celular en 4 ml de HBSS que contiene 1 mg / ml de inhibidor a base de soja de la enzima. Pipetear suavemente varias veces con un 1 ml de pipette para obtener una suspensión de células individuales. </li><li> Células piscina de los dos compendios o alternativamente mantienen digiere celulares separados hasta después de los recuentos de células. NOTA: Esto evita la mezcla de células potencialmente bajas de viabilidad con pilas de gran viabilidad. </li><li> Añadir HBSS a un volumen final de 10-20 ml y llevar a cabo el recuento de células usando azul de tripano para medir la viabilidad. </li><li> Confirme el fenotipo de las células epiteliales mediante citometría de flujo. Invente cócteles de anticuerpos adecuados utilizando anti-EpCAM-PE (dilución 1: 2) anti-CD90-PECy5 (1: 250) y anti-CD105-APC (1: 100) en HBSS. </li><li> Resuspender un subconjunto de células de uno o ambos digiere en el cóctel de anticuerpos a una concentración de 25 x 10 6 células / ml. </li><li> Para citometría de flujo, la mancha 1 x 10 6 células con los cócteles de anticuerpos que contienen controles de isotipo (por ejemplo. El control-PE isotipo IgG1 + anti-CD90-PECy5 + anti-CD105-APC). Mantenga ~ 1 x 10 6 células sin teñir para citómetro de flujo de configuración. </li><li> Se incuban las células en anticuerpocócteles a 4 ° C durante 60 minutos. </li><li> Lavar las células 3 veces en HBSS y se resuspende en 5 a 10 millones de células por ml para citometría de flujo. </li><li> Realizar citometría de flujo y los resultados de nota. Se espera que la pureza de la cepa aislada de células (el porcentaje de EpCAM positivo y CD90 / CD105 células negativas) para ser 90-95% y &lt;1%, respectivamente. </li><li> Ponga a un lado un número adecuado de células para la prueba de los criterios de control de calidad o de liberación, según lo determinemos aplicación final. NOTA: Esto puede incluir la enfermedad / detección viral u otra seguridad o criterios biológicos. </li><li> Para las células restantes, siga el protocolo de crioconservación, o alternativamente colocar directamente en la cultura (paso directo a la cultura 3.6). </li></ol> 2. Criopreservación de HAECs <ol style="margin-left: 40px;"><li> Determinar el número de células y la viabilidad mediante citometría de flujo de datos o el conteo manual con exclusión de azul tripán. </li><li> Centrifugar a 700 g durante 5 min. </li><li> Resuspender celulars entre 1 hasta 10 millones de células por ml en animales medios criopreservación-producto libre. </li><li> Verter un volumen adecuado de las células en viales de crioconservación O-anillos y coloque en un aparato de congelación velocidad controlada, donde se produce el enfriamiento a aproximadamente 1 ° C / min hasta -80 ° C se logra. </li><li> Transferencia viales en nitrógeno líquido para almacenamiento a largo plazo. </li></ol> 3. La descongelación y Cultura de Cyropreserved HAECs <ol style="margin-left: 40px;"><li> Retire los viales de crioconservación de nitrógeno líquido y colocar en hielo. NOTA: Para transferir solamente - proceder rápidamente a la siguiente etapa. </li><li> Descongele los viales de crioconservación rápidamente a 37 ° C hasta que sólo una pequeña parte (~ 5 mm x 5 mm) de los restos de hielo. NOTA: Es importante que la solución de células no se calienta para reducir los efectos tóxicos de DMSO en los medios de crioconservación. </li><li> Diluir los medios de crioconservación 10 veces con frío (~ 4 ° C) medios libres de suero, añadido gota a gota en un tamaño adecuadod tubo o contenedor. </li><li> Centrifugar a 700 xg durante 5 min a TA. </li><li> Resuspender en medio libre de suero, a una concentración aproximada de 1 millón de células por ml. Determinar el número de células y la viabilidad mediante conteo automatizado o manual con exclusión de azul tripán. Esperado viabilidad después de la descongelación será un 5-10% menos de viabilidad antes de la criopreservación. </li><li> Células de la placa de cerámica de plástico de cultivo tratadas con células estándar, o bien, utilizar colágeno de tipo I a las superficies de cultivo de abrigo. </li><li> Por tipo I revestimiento colágeno seguir el protocolo de abajo <ol><li> Preparar ácido soluble tipo I colágeno de placenta humana (colágeno de 15 mg con 25 ml de agua desionizada y 50 ácido acético glacial l). </li><li> Cubra vaso con Parafilm y una agitación moderada a 37 ° C hasta que se disuelven fibras de colágeno. </li><li> Solución de colágeno filtro estéril utilizando un filtro de 0,22 micras. </li><li> Diluir el colágeno Stock 01:10 filtrada con agua destilada. Esta acción diluida es la conce trabajosolución ntration para superficies de plástico de recubrimiento y de membrana. </li><li> Plástico capa de colágeno, las superficies de cristal y de membrana durante 2 horas a temperatura ambiente a 37 ° C, o O / N a 2-8 ° C. </li><li> Retire el exceso de líquido de la superficie recubierta, y deje que se seque O / N. </li><li> Enjuague con agua de grado de cultivo de tejido estéril o HBSS antes de la introducción de células y el medio. </li></ol></li><li> HAECs placa a una densidad de 25.000 células / cm 2 en medio libre de suero, y permiten la conexión que se produzca durante 72-96 horas. </li><li> Después de la unión, cambiar el medio por aspiración con cuidado y reemplazar con un volumen adecuado de medio libre de suero cada 2-3 días. </li><li> Passage células utilizando el protocolo de abajo <ol><li> Aspirar los medios de cultivo celular y lavar con un volumen de 1x estéril HBSS </li><li> Añadir volumen 1x de la solución de análisis enzimático e incubar durante 5 a 15 minutos, o hasta que las células se observaron separe de la superficie de cultivo de células. </li><li> Recoger digerido enzimático solución que contiene células, asegurando ceLLS se desprenden de la superficie de cultivo de células pipeteando repetidamente la solución enzimática digerir contra la superficie de cultivo de células </li><li> Centrifugar a 700 g durante 5 min. </li><li> Resuspender el sedimento celular en un volumen adecuado de HBSS que contiene 1 mg / ml de inhibidor a base de soja enzima. Pipetear suavemente varias veces con una pipeta de 1 ml para obtener una suspensión de células individuales. </li><li> Añadir medio libre de suero a una concentración aproximada de 1 millón de células / ml. Determinar el número de células y la viabilidad mediante conteo automatizado o manual con exclusión de azul tripán. </li><li> HAECs placa a una densidad de 25.000 células / cm 2 en medio libre de suero, y continúan con los métodos estándar de cultivo. </li></ol></li></ol>

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The authors have nothing to disclose.

Hay varios parámetros críticos que pueden tener un impacto significativo en el éxito de esta metodología. El almacenamiento de la placenta o el amnios para hasta 3 horas antes del aislamiento de HAECs puede ser deseable para fines logísticos o de programación, sin embargo se recomienda que el tejido se procesa tan pronto como sea posible. Si el tejido se va a almacenar, se recomienda que se realice el almacenamiento después de la disección y el lavado de la membrana amnios. Amnion se puede almacenar en HBSS que ...

Las células derivadas de fuentes perinatales, como la placenta, membranas de la placenta, el cordón umbilical y el líquido amniótico han atraído la atención de investigadores y clínicos como una fuente potencial de células para la medicina regenerativa 1,2. La razón de este interés es que todos estos tipos de células poseen un cierto grado de plasticidad y capacidad inmunomoduladora 3, propiedades que son fundamentales para sus aplicaciones terapéuticas potenciales. HAECs son una población heterogénea epitelial que se puede derivar de plazo o pre-término membrana amnios 4, proporcionando una abundante fuente potencial de material celular regenerativa. Las propiedades que hacen que HAECs atractivos como una terapia celular incluyen su multipotencia, baja inmunogenicidad, y las propiedades anti-inflamatorias. HAECs se han encontrado para ser altamente multipotentes tanto in vitro como in vivo, capaces de diferenciarse en linajes mesodérmicos (cardiomyocytes, miocitos, adipocitos, osteocitos), de origen endodérmico (células pancreáticas, células hepáticas, células pulmonares) y linajes ectodérmicas (pelo, piel, células neuronales y astrocitos) 5-10. De modo tranquilizador, a pesar de sus HAECs multipotencialidad no parecen bien forman tumores o promueven el desarrollo de tumores in vivo. Además, HAECs también son inmunes privilegiados, que expresa bajos niveles de antígenos de clase II leucocitarios humanos (HLA) 8. Esta propiedad probablemente subyace en su capacidad para evadir el rechazo inmunológico después del trasplante alogénico y xenogénico, como se demuestra en los estudios que utilizaron monos inmunes competentes, conejos, cobayas, ratas y cerdos 11-13. HAECs mostrar inmunomodulador potentes propiedades inmunosupresoras y por lo tanto ofrecen ventajas prácticas significativas para potenciales aplicaciones clínicas en el tratamiento de la enfermedad autoinmune. HAECs se cree que ejercen funciones inmunomoduladoras tanto en el sistema inmune innata y adaptativa. Ene de los mecanismos sugeridos, es a través de la secreción de factores inmunomoduladores 14. Las aplicaciones actuales de HAECs en modelos de enfermedades de animales pre-clínicos incluyen el tratamiento de apoplejía, esclerosis múltiple, enfermedad hepática, diabetes y enfermedades pulmonares crónicas y agudas. Los investigadores han mostrado interés en utilizar HAECs para tratar posterior al accidente cerebrovascular inflamación cerebral debido a sus propiedades únicas. Hay evidencia de que HAECs puede cruzar la barrera de sangre del cerebro donde pueden injertarse, sobrevivir durante hasta 60 días, de diferenciarse en neuronas, disminuir la inflamación y promover la regeneración del tejido nervioso central dañado sistema en modelos animales de enfermedades neurológicas 15. HAECs ofrecen la posibilidad de dirigir e invertir múltiples vías patológicos que contribuyen al desarrollo y la progresión de la esclerosis múltiple. Por ejemplo, los resultados de estudios en animales pre-clínicos sugieren que HAECs son fuertemente inmunosupresora ypuede potencialmente inducir tolerancia inmune periférica y revertir las respuestas inflamatorias en curso. HAECs también se han demostrado tener la capacidad de diferenciarse en células neuronales in vivo y mejorar la neuroregeneración endógena a través de la secreción de una amplia gama de factores neurotróficos 16. Células epiteliales humanas y amnios roedor ya han demostrado su eficacia terapéutica para el tratamiento de la enfermedad hepática en modelos animales. En un modelo de daño de inducción tetracloruro de carbono de la enfermedad hepática, haec trasplante de plomo para el injerto de HAECs viables en el hígado, acompañada con una reducción de la apoptosis de hepatocitos, y la disminución de la inflamación y la fibrosis hepática 17. HAECs se puede estimular a los factores de páncreas expresadas incluyendo insulina y glucosa transportistas. Varios estudios han investigado el potencial de HAECs para restablecer los niveles de glucosa en sangre en ratones diabéticos 18. En los ratones que recibieronHAECs, tanto a nivel de peso y glucosa en la sangre del cuerpo de los animales disminuyó a niveles normales después de la inyección de las células. Estos estudios presentan un caso fuerte para el uso de HAECs para el tratamiento de la diabetes mellitus. HAECs tienen un papel demostrado en la prevención y reparación de la lesión pulmonar aguda y crónica experimental, tanto en adultos y neonatales 19 modelos. Estos estudios encontraron que HAECs diferencian in vitro en células epiteliales del pulmón funcional que expresan múltiples proteínas pulmonares asociadas, incluyendo la fibrosis quística regulador de conductancia transmembrana (CFTR), el canal iónico que está mutado en pacientes con fibrosis quística 20. Además, cuando HAECs se entregan al adulto lesionado y pulmonar neonatal, que ejercen sus efectos a través de la modulación de reparación de las células inmunitarias del huésped, reduciendo el reclutamiento de leucocitos pulmonar, incluyendo neutrófilos, macrófagos y linfocitos 21-23. Dada su abundancia,historial de seguridad y aplicaciones clínicas probadas para varias enfermedades, los ensayos clínicos utilizando HAECs es inevitable. Con el objetivo de acelerar la traducción de terapias HAEC a de ensayos clínicos, hemos desarrollado métodos para aislar, criopreservar y HAECs cultura de una manera adecuada para los ensayos clínicos, el uso de reactivos a productos libres de animales de acuerdo con las buenas prácticas de fabricación actuales (cGMP) directrices . Basamos este protocolo un protocolo previamente publicado que estábamos usando con éxito para aislar HAECs utilizando reactivos de origen animal 6. Hemos modificado el protocolo original para reemplazar productos de origen animal con reactivos de productos libre de animales, y la optimización posterior se realizó para optimizar el rendimiento de células, la viabilidad y pureza. Nuestro objetivo era desarrollar un protocolo que cumplir con las normas reglamentarias para la fabricación de células para los ensayos clínicos en humanos.

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			<td height="21" style="height:21px;width:220px;">Name of Material/ Equipment</td>
			<td style="width:119px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:184px;">Comments/Description</td>
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			<td height="21" style="height:21px;width:220px;">Collection Kit</td>
			<td style="width:119px;"></td>
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			<td></td>
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			<td height="21" style="height:21px;width:220px;">Stripping tray</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td style="width:119px;">13-361B</td>
			<td></td>
		</tr>
		<tr height="34">
			<td height="34" style="height:34px;width:220px;">Liberty Dressing Forcep, pointed 13cm</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>S17329&#160;</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Scissors - Sharp/Blunt Straight</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>NC0562592&#160;</td>
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			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
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			<td height="21" style="height:21px;width:220px;">Protective Apparel (Gown)</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
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			<td height="21" style="height:21px;">Protective Apparel</td>
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			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">Isolation gowns</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-15374-M</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sterile latex gloves&#160;</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>19-014-643&#160;</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">General purpose face mask</td>
			<td style="width:119px;">Cardinal Health</td>
			<td style="width:119px;">AT7511</td>
			<td></td>
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			<td height="21" style="height:21px;">Bonnets</td>
			<td style="width:119px;">Medline</td>
			<td style="width:119px;">CRI1001</td>
			<td></td>
		</tr>
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			<td height="21" style="height:21px;">Shoe covers</td>
			<td style="width:119px;">U-line</td>
			<td style="width:119px;">S-7873W</td>
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			<td height="21" style="height:21px;width:220px;"></td>
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			<td height="21" style="height:21px;">Media and Reagents</td>
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			<td style="width:119px;"></td>
			<td></td>
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			<td height="33" style="height:33px;">Hanks&#8217; Balanced Salt Solution (HBSS)</td>
			<td style="width:119px;">Life Technologies</td>
			<td align="right" style="width:119px;">14175095</td>
			<td>without calcium or magnesium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TrypZean(animal product&#8211;free recombinant trypsin)</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T3449</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Soybean Trypsin Inhibitor&#160; 1g/50mL</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td style="width:119px;">T6522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cryostor CS5</td>
			<td style="width:119px;">BioLife Solutions</td>
			<td align="right" style="width:119px;">205102</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Trypan blue reagent</td>
			<td style="width:119px;">Life Technologies</td>
			<td style="width:119px;">15250-061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-EpCam-PE</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130 - 091-253</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PE-isotype control</td>
			<td style="width:119px;">Miltenyi Biotec</td>
			<td style="width:119px;">130-098-845</td>
			<td></td>
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			<td height="21" style="height:21px;">anti-CD90-PeCy5</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">555597</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;">PeCy5-isotype control</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">557224</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">anti-CD105-APC</td>
			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">562408</td>
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		<tr height="21">
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			<td style="width:119px;">BD Pharmingen</td>
			<td align="right" style="width:119px;">340754</td>
			<td></td>
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			<td height="21" style="height:21px;">Collagen Type VI</td>
			<td style="width:119px;">Sigma Aldrich</td>
			<td>C7521</td>
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			<td height="21" style="height:21px;">50mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356550</td>
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		<tr height="21">
			<td height="21" style="height:21px;">10mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356551</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5mL graduated pipette</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>356543</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15mL falcon tubes</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352096</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">15-cm petri dishes</td>
			<td style="width:119px;">Corning</td>
			<td align="right" style="width:119px;">351058</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">70-&#956;m filters</td>
			<td style="width:119px;">BD/Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">0.22-&#956;m filters</td>
			<td style="width:119px;">Millipore</td>
			<td>SLGV033RS</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">1ml Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-401</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">200ul Pipette tips</td>
			<td style="width:119px;">Fisherbrand</td>
			<td style="width:119px;">02-707-409</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">20ml Syringe</td>
			<td style="width:119px;">BD/Medical</td>
			<td align="right" style="width:119px;">309661</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic spatula</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>14-245-97&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Plastic weighing boat</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>02-202-102&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Cryo vials</td>
			<td>Nunc</td>
			<td>377267</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:220px;"></td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Equipment</td>
			<td style="width:119px;"></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Mr Frosty</td>
			<td style="width:119px;">Fisher Scientific</td>
			<td>A451-4&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:220px;">Biohazard Cabinet</td>
			<td style="width:119px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

isolation, cryopreservation and culture of human amnion epithelial cells for clinical applications

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a potential source of cells for regenerative medicine. The reason for this interest is evidence that these cells have highly multipotent differentiation ability, low immunogenicity, and anti-inflammatory functions. These properties have prompted researchers to investigate the potential of hAECs to be used to treat a variety of diseases and disorders in pre-clinical animal studies with much success.
hAECs have found widespread application for the treatment of a range of diseases and disorders. Potential clinical applications of hAECs include the treatment of stroke, multiple sclerosis, liver disease, diabetes and chronic and acute lung diseases. Progressing from pre-clinical animal studies into clinical trials requires a higher standard of quality control and safety for cell therapy products. For safety and quality control considerations, it is preferred that cell isolation protocols use animal product-free reagents. 
We have developed protocols to allow researchers to isolate, cryopreserve and culture hAECs using animal product-free reagents. The advantage of this method is that these cells can be isolated, characterized, cryopreserved and cultured without the risk of delivering potentially harmful animal pathogens to humans, while maintaining suitable cell yields, viabilities and growth potential. For researchers moving from pre-clinical animal studies to clinical trials, these methodologies will greatly accelerate regulatory approval, decrease risks and improve the quality of their therapeutic cell population.

Cuando este procedimiento se sigue correctamente, se debe esperar un rendimiento promedio de 120 millones de HAECs, con un rango típico de 80 hasta 160 millones de células. A partir de estos rendimientos, una viabilidad media de 83 ± 4% se puede esperar. El aumento de rendimiento medio y la viabilidad ligeramente inferior en el método clínico puede ser debido a la mayor actividad de la tripsina que el producto derivado de un animal, y quizás también debido a la falta de proteínas séricas. HAECs aisladas tienen ...

Watch this Scientific Journal Video about Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications at JoVE.com

Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines.

El aislamiento, la criopreservación y cultivo de células epiteliales Amnion Humanos para Aplicaciones clínicas

Human amnion epithelial cells (hAECs) derived from term or pre-term amnion membranes have attracted attention from researchers and clinicians as a ...

isolation-cryopreservation-culture-human-amnion-epithelial-cells-for

Research

JoVE Journal

Medicine

13.8K vistas.  Wake Forest University Health Sciences. We describe a protocol to isolate and culture human amnion epithelial cells (hAECs) using animal product-free reagents in accordance with current good manufacturing practices (cGMP) guidelines. 

Video: Isolation, Cryopreservation and Culture of Human Amnion Epithelial Cells for Clinical Applications

Isolation, Characterization and Comparative Differentiation of Human Dental Pulp Stem Cells Derived from Permanent Teeth by Using Two Different Methods

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human pulp-derived stem cells (hDPSCs) possess high proliferation potential with multi-lineage differentiation capacity compare to the ordinary source of adult stem cells1-8; therefore, hDPSCs could be the good candidates for autologous transplantation in tissue engineering and regenerative medicine. Along with these benefits, possessing the mesenchymal stem cells (MSC) features, such as immunolodulatory effect, make hDPSCs more valuable, even in the case of allograft transplantation6,9,10. Therefore, the primary step for using this source of stem cells is to select the best protocol for isolating hDPSCs from pulp tissue. In order to achieve this goal, it is crucial to investigate the effect of various isolation conditions on different cellular behaviors, such as their common surface markers &amp; also their differentiation capacity.
Thus, here we separate human pulp tissue from impacted third molar teeth, and then used both existing protocols based on literature, for isolating hDPSCs,11-13 i.e. enzymatic dissociation of pulp tissue (DPSC-ED) or outgrowth from tissue explants (DPSC-OG). In this regards, we tried to facilitate the isolation methods by using dental diamond disk. Then, these cells characterized in terms of stromal-associated Markers (CD73, CD90, CD105 &amp; CD44), hematopoietic/endothelial Markers (CD34, CD45 &amp; CD11b), perivascular marker, like CD146 and also STRO-1. Afterwards, these two protocols were compared based on the differentiation potency into odontoblasts by both quantitative polymerase chain reaction (QPCR) &amp; Alizarin Red Staining. QPCR were used for the assessment of the expression of the mineralization-related genes (alkaline phosphatase; ALP, matrix extracellular phosphoglycoprotein; MEPE &amp; dentin sialophosphoprotein; DSPP).14

The method described isolation and characterization of human Dental Pulp Stem Cells (hDPSCs) by using either enzymatic dissociation of pulp (DPSC-ED) or direct outgrowth of stem cells from pulp tissue explants (DPSC-OG). Then followed by in vitro comparative differentiation of both types of hDPSCs into odontoblasts.

Diferenciación de aislamiento, caracterización y comparativo de las células madre derivadas de la Pulpa Dental de los dientes permanentes mediante dos métodos diferentes

Developing wisdom teeth are easy-accessible source of stem cells during the adulthood which could be obtained by routine orthodontic treatments. Human ...

Investigación

Medicina

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.
Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.
Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133+ CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133+ CSCs and CD133- bulk, which can be cultivated and functionally analyzed thereafter.

Patient Derived Cell Culture and Isolation of CD133+ Putative Cancer Stem Cells from Melanoma

This article describes the preparation of freshly obtained melanoma tissue into primary cell cultures, and how to remove contaminations of erythrocytes and fibroblasts from the tumor cells. Finally, we describe how CD133+ putative melanoma stem cells are sorted from the CD133- bulk using Magnetic Activated Cell Sorting (MACS).

Paciente celulares derivadas Cultura y aislamiento de CD133<sup&gt; +</sup&gt; Putativos células madre del cáncer de melanoma

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for ...

Isolation, Culture, and Imaging of Human Fetal Pancreatic Cell Clusters

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning endocrine cells, as evidenced by a significant increase in circulating human C-peptide following glucose stimulation1-9. However in vitro, genesis of insulin producing cells from human fetal ICCs is low10; results reminiscent of recent experiments performed with human embryonic stem cells (hESC), a renewable source of cells that hold great promise as a potential therapeutic treatment for type 1 diabetes. Like ICCs, transplantation of partially differentiated hESC generate glucose responsive, insulin producing cells, but in vitro genesis of insulin producing cells from hESC is much less robust11-17. A complete understanding of the factors that influence the growth and differentiation of endocrine precursor cells will likely require data generated from both ICCs and hESC. While a number of protocols exist to generate insulin producing cells from hESC in vitro11-22, far fewer exist for ICCs10,23,24. Part of that discrepancy likely comes from the difficulty of working with human fetal pancreas. Towards that end, we have continued to build upon existing methods to isolate fetal islets from human pancreases with gestational ages ranging from 12 to 23 weeks, grow the cells as a monolayer or in suspension, and image for cell proliferation, pancreatic markers and human hormones including glucagon and C-peptide. ICCs generated by the protocol described below result in C-peptide release after transplantation under the kidney capsule of nude mice that are similar to C-peptide levels obtained by transplantation of fresh tissue6. Although the examples presented here focus upon the pancreatic endoderm proliferation and &#946;&#160;cell genesis, the protocol can be employed to study other aspects of pancreatic development, including exocrine, ductal, and other hormone producing cells.

A protocol to isolate, culture, and image islet cell clusters (ICCs) derived from human fetal pancreatic cells is described.&#160; The method details the steps necessary to generate ICCs from tissue, culture as monolayers or in suspension as aggregates, and image for markers of proliferation and pancreatic cell fate decisions.

El aislamiento, la cultura, y de imágenes de Human Fetal pancreáticas grupos de células

For almost 30 years, scientists have demonstrated that human fetal ICCs transplanted under the kidney capsule of nude mice matured into functioning ...

Adaptation of Semiautomated Circulating Tumor Cell (CTC) Assays for Clinical and Preclinical Research Applications

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the presence of circulating tumor cells (CTCs). These rare cells have been demonstrated to be of clinical significance in metastatic breast, prostate, and colorectal cancers. The current gold standard in clinical CTC detection and enumeration is the FDA-cleared CellSearch system (CSS). This manuscript outlines the standard protocol utilized by this platform as well as two&#160;additional adapted protocols that describe the detailed process of user-defined marker optimization for protein characterization of patient CTCs and a comparable protocol for CTC capture in very low volumes of blood, using standard CSS reagents, for studying in vivo preclinical mouse models of metastasis. In addition, differences in CTC quality between healthy donor blood spiked with cells from tissue culture versus patient blood samples are highlighted. Finally, several commonly discrepant items that can lead to CTC misclassification errors are outlined. Taken together, these protocols will provide a useful resource for users of this platform interested in preclinical and clinical research pertaining to metastasis and CTCs.

Adaptation of Semiautomated Circulating Tumor Cell CTC Assays for Clinical and Preclinical Research Applications

Circulating tumor cells (CTCs) are prognostic in several metastatic cancers. This manuscript describes the gold standard CellSearch system (CSS) CTC enumeration platform and highlights common misclassification errors. In addition, two&#160;adapted protocols are described for user-defined marker characterization of CTCs and CTC enumeration in preclinical mouse models of metastasis using this technology.

Adaptación de semiautomatizado de células tumorales circulantes (CTC) Ensayos para aplicaciones clínicas y de investigación preclínica

The majority of cancer-related deaths occur subsequent to the development of metastatic disease. This highly lethal disease stage is associated with the ...

Isolation and Culture Expansion of Tumor-specific Endothelial Cells

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model for developing new angiogenesis inhibitors for cancer. However, long-term in vitro expansion of murine endothelial cells (EC) is challenging due to phenotypic drift in culture (endothelial-to-mesenchymal transition) and contamination with non-EC. This is especially true for TEC which are readily outcompeted by co-purified fibroblasts or tumor cells in culture. Here, a high fidelity isolation method that takes advantage of immunomagnetic enrichment coupled with colony selection and in vitro expansion is described. This approach generates pure EC fractions that are entirely free of contaminating stromal or tumor cells. It is also shown that lineage-traced Cdh5cre:ZsGreenl/s/l reporter mice, used with the protocol described herein, are a valuable tool to verify cell purity as the isolated EC colonies from these mice show durable and brilliant ZsGreen fluorescence in culture.

We report a reliable method to isolate and culture primary tumor-specific endothelial cells from genetically engineered mouse models.

Aislamiento y cultivo de expansión de las células endoteliales-tumorales específicas

Freshly isolated tumor-specific endothelial cells (TEC) can be used to explore molecular mechanisms of tumor angiogenesis and serve as an in vitro model ...

Three-dimensional Alginate-bead Culture of Human Pituitary Adenoma Cells

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from brown sea algae. Briefly, the tumor tissue is cut into small pieces and submitted to an enzymatic digestion with collagenase and trypsin. Next, a cell suspension is obtained. The tumor cell suspension is mixed with 1.2% sodium alginate and dropped into a CaCl2 solution, and the alginate/cell suspension is gelled on contact with the CaCl2 to form spherical beads. The cells embedded in the alginate beads are supplied with nutrients provided by the culture media enriched with 20% FBS. Three-dimensional culture in alginate beads maintains the viability of adenoma cells for long periods of time, up to four months. Moreover, the cells can be liberated from the alginate by washing the beads with sodium citrate and seeded on glass coverslips for further immunocytochemical analyses. The use of a cell culture model allows for the fixation and visualization of the actin cytoskeleton with minimal disorganization. In summary, alginate beads provide a reliable culture system for the maintenance of pituitary adenoma cells.

Three-dimensional culture in alginate beads, due to its simplicity and reproducibility, was chosen to maintain the pituitary adenoma cells. The procedures included an initial enzymatic digestion and mechanical dissociation of the tumor tissue, and the subsequent cell suspension was encapsulated in alginate beads.

Tridimensional de alginato-grano Cultura de células humanas del adenoma pituitario

A three-dimensional culture method is described in which primary pituitary adenoma cells are grown in alginate beads. Alginate is a polymer derived from ...

Isolation and Culture of Primary Endothelial Cells from Canine Arteries and Veins

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study vasculogenesis, (tumor) angiogenesis, and atherosclerosis. The current standard for in vitro research on human endothelial cells (ECs) is the use of Human Umbilical Vein Endothelial Cells (HUVECs) and Human Umbilical Artery Endothelial Cells (HUAECs). For canine endothelial research, only one cell line (CnAOEC) is available, which is derived from canine aortic endothelium. Although currently not completely understood, there is a difference between ECs originating from either arteries or veins. For a more direct approach to in vitro functionality studies on ECs, we describe a new method for isolating Canine Primary Endothelial Cells (CaPECs) from a variety of vessels. This technique reduces the chance of contamination with fast-growing cells such as fibroblasts and smooth muscle cells, a problem that is common in standard isolation methods such as flushing the vessel with enzymatic solutions or mincing the vessel prior to digestion of the tissue containing all cells. The technique we describe was optimized for the canine model, but can easily be utilized in other species such as human.

Novel isolation methods of primary endothelial cells from blood vessels are needed. This protocol describes a new technique that completely inverts blood vessels of interest, exposing only the endothelial side to enzymatic digestion. The resulting pure endothelial cell culture can be used to study cardiovascular diseases, disease modelling, and angiogenesis.

Aislamiento y cultivo de células endoteliales primarias de las arterias y venas caninas

Cardiovascular disease is studied in both human and veterinary medicine. Endothelial cells have been used extensively as an in vitro model to study ...

A Novel Clinical Grade Isolation Method for Human Kidney Perivascular Stromal Cells

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular Stromal Cells (PSCs) share characteristics with bone marrow MSCs (bmMSCs). However, they also possess, most likely due to local imprinting, tissue-specific properties and play a role in local tissue homeostasis. This tissue specificity may result in tissue specific repair, also within the human kidney. We previously showed that human kidney PSCs (kPSCs) have enhanced kidney epithelial wound healing whereas bmMSCs did not have this potential. Moreover, kPSCs can ameliorate kidney injury in vivo. Therefore, kPSCs constitute an interesting source for cell therapy, particularly for kidney diseases and renal transplantation. Here we show the detailed isolation and culture method for kPSCs from transplant-grade human kidneys based on whole-organ perfusion of digestive enzymes via the renal artery and enrichment for the perivascular marker NG2. In this way, large cell quantities can be obtained that are suitable for cellular therapy.

Here we present a novel clinical grade isolation and culture method for kidney Perivascular Stromal Cells (kPSCs) based on whole organ perfusion with digestive enzymes and NG2-cell enrichment. With this method, it is possible to acquire sufficient cell numbers for cellular therapy.

Un método de aislamiento de grado clínico novedoso para Stromal células de riñón humano perivasculares

Mesenchymal Stromal Cells (MSCs) are tissue homeostatic and immune modulatory cells that have shown beneficial effects in kidney diseases and ...

Culture and Imaging of Human Nasal Epithelial Organoids

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis Transmembrane conductance Regulator (CFTR) activity and restoration from small molecule compounds. Our group recently focused on establishing a well-differentiated organoid model directly derived from primary human nasal epithelial cells (HNE). Histology of sectioned organoids, whole-mount immunofluorescent staining, and imaging (using confocal microscopy, immunofluorescent microscopy, and bright field) are essential to characterize organoids and confirm epithelial differentiation in preparation for functional assays. Furthermore, HNE organoids produce lumens of varying sizes that correlate with CFTR activity, distinguishing between CF and non-CF organoids. In this manuscript, the methodology for culturing HNE organoids are described in detail, focusing on the assessment of differentiation using the imaging modalities, including the measurement of baseline lumen area (a method of CFTR activity measurement in organoids that any laboratory with a microscope can employ) as well as the developed automated approach to a functional assay (which requires more specialized equipment).

A detailed protocol is presented here to describe an in vitro organoid model from human nasal epithelial cells. The protocol has options for measurements requiring standard laboratory equipment, with additional possibilities for specialized equipment and software.

Cultivo e imágenes de organoides epiteliales nasales humanos

Individualized therapy for cystic fibrosis (CF) patients can be achieved with an in vitro disease model to understand baseline Cystic Fibrosis ...

Isolation and Culture of Human Adipose-Derived Stem Cells With an Innovative Xenogeneic-Free Method for Human Therapy

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission due to the introduction of animal-derived products during in vitro manipulation. The xenogeneic components, such as collagenase or fetal bovine serum, commonly used during the cell isolation and expansion steps could be associated with the potential risks of immune reactivity or viral, bacterial, and prion infection in the receiving patients. Following good manufacturing practice guidelines, chemical tissue dissociation should be avoided, while fetal bovine serum (FBS) can be substituted with xenogeneic-free supplements. Moreover, to ensure the safety of cell products, the definition of more reliable and reproducible methods is important. We have developed an innovative, completely xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells without altering their properties compared to collagenase FBS-cultured standard protocols. Here, human adipose-derived stem cells (hASCs) were isolated from abdominal adipose tissue. The sample was mechanically minced with scissors/a scalpel, micro-dissected and mechanically dispersed in a 10 cm Petri dish, and prepared with scalpel incisions to facilitate the attachment of the tissue fragments and the migration of hASCs. Following the washing steps, hASCs were selected due to their plastic adherence without enzymatic digestion. The isolated hASCs were cultured with medium supplemented with 5% heparin-free human platelet lysate and detached with an animal-free trypsin substitute. Following good manufacturing practice (GMP) directions on the production of cell products intended for human therapy, no antibiotics were used in any culture media.

Xenogeneic (chemical or animal-derived) products introduced in the cell therapy preparation/manipulation steps are associated with an increased risk of immune reactivity and pathogenic transmission in host patients. Here, a complete xenogeneic-free method for the isolation and in vitro expansion of human adipose-derived stem cells is described.

Aislamiento y cultivo de células madre derivadas de tejido adiposo humano con un innovador método libre de xenogénicos para terapia humana

Considering the increasing impact of stem cell therapy, biosafety concerns have been raised regarding potential contamination or infection transmission ...