Soporte: NIDCD R01DC009801, P30DC010362, 5T32DC000040-17

Declaración de Ética: el uso de las muestras de tumores humanos en este protocolo fue aprobado por la Universidad de Iowa Junta de Revisión Institucional (IRB). 1. Configuración del día de antes de la cosecha del tumor <ol><li> Placas de cultivo Escudo celulares: En una campana de cultivo estéril, añadir una cantidad suficiente (solución al 0,01%) de poli-L-ornitina para cubrir el fondo de una cultura de poliestireno / plástico y / plato, reemplace las tapas, y deje reposar a temperatura ambiente durante al menos 2 hr. Los portaobjetos de vidrio o cubreobjetos pueden ser sustituidos por poliestireno para los experimentos que requieren imágenes. </li><li> Después de 2 horas, retire el-L-ornitina poli solución con succión y permiten que las placas se sequen por completo en unas capuchas de cultivo estériles. Añadir la solución de laminina suficiente (10 g / ml de Ca 2 + / Mg 2 + libre de HBSS [HBSS-/ -]) para cubrir completamente las placas de cultivo, reemplace las tapas, a continuación, ya sea 1) lugar en fresco 4 ° C refrigerador durante la noche, o 2) colocar en 37 ° C incubadora durante al menos 2 h. Si las placasse almacenará toda la noche o más tiempo, envuelva las placas en una envoltura de plástico para evitar la evaporación / desecación. </li></ol> 2. Configuración del Día del Tumor Harvest <ol><li> Preparar Cultura Medios: Medios es recién hecho en el día de la cosecha y el procesamiento del tumor, se almacena a 4 ° C, y se calienta a 37 ° C en una incubadora inmediatamente antes de los medios de comunicación adiciones / cambios. </li><li> Medios de cultivo Schwannoma es de alta glucosa (4,5 mg / ml) de DMEM (con rojo de fenol) con 0,1 mg / ml de penicilina y 0,1 mg / ml de estreptomicina; Medios N2 complementan dilución final 1:100; Insulina bovina 5 g / ml; De suero fetal bovino al volumen total del 10%. Filtrar todos los medios de comunicación a través de filtro de 0,22 micras. </li><li> Preparar Transporte de muestras del refrigerador: Enviar un refrigerador lleno de hielo con 1-2 (dependiendo de la cantidad de tumor que se recogerá a) 50 ml tubos cónicos estériles con 25 ml de HBSS con Ca 2 + / Mg 2 + (HBSS + / +) a la sala de operaciones para la transferencia de la muestra del tumor y el transporte. </li><li> Preparar indivtubos de disociación tumor iDual - llenan estériles de 2 ml tubos redondos de fondo con 1 ml de HBSS + / + y colocar en hielo. (Tubos será utilizado para disección aguda / disociación de muestras de tumor). </li><li> Para la disociación, preparar aproximadamente un tubo de disociación para cada 0,5 cm 3 de tejido recogido; Por ejemplo, una muestra de tumor de 1,0 x 1,0 x 1,0 cm (1 cm 3) requerirán dos tubos de + / + de disociación de HBSS. </li><li> La luz ultravioleta de esterilización: Tijeras Place tejido, pinzas pequeñas, mango de bisturí, 1x plato normal de Petri, P1000 pipeta y puntas de pipeta en una campana de cultivo estéril y dejar a la luz ultravioleta durante aproximadamente 15 minutos antes de la llegada y el procesamiento de tejido tumoral. </li></ol> 3. Aislamiento de muestras y transporte <ol><li> Aislar las muestras de tumor mediante resección quirúrgica. </li><li> Colocar inmediatamente las muestras de tumor en el HBSS enfriado con hielo + / + tan rápidamente como sea posible después de retirarlos del paciente. Una vez que todos tissuSe recogen muestras de correos, el transporte de muestras (en hielo) desde el teatro operativo al laboratorio para su posterior procesamiento. </li></ol> 4. La disociación de tejidos y la trituración <ol><li> Siguiendo una técnica estéril estándar en una campana de flujo laminar, traiga tubo cónico con muestras tumorales en la campana de cultivo, y lavar las muestras por tubo y tumor 50x invirtiendo. Permitir fragmentos caigan a la parte inferior del tubo, y luego retire HBSS + / +. Vuelva a llenarlo con 30 ml de HBSS + / +, y agitar / 50x invertir de nuevo. Repetir la extracción de inversión / media para un total de 4 veces. </li><li> Vuelva a suspender fragmentos de tumor en 30 ml de HBSS + / +, por última vez, a continuación, vierta la mezcla en un plato de petri mm sterile100 y tumorales separados en 0,5 cm 3 agrupaciones. Si se encuentran fragmentos de tumor más grandes, use las tijeras de tejidos o bisturí (# 11 o # 15 cuchillas) para cortar en pedazos más pequeños. </li><li> Utilice las pinzas para colocar los 0,5 cm 3 grupos de tejidos en el individuo HBSS + / + lleno, 2 ml de fondo redondo cónicatubos al, todo el hielo. Utilice las tijeras de tejidos para picar fragmentos de tumor en sub-1 mm fragmentos-la suspensión debe tener un aspecto &#39;globo de nieve&#39; (Figura 1). Picar el tumor sólo hasta que la mayoría de los fragmentos son sub-1 mm; no &quot;sobre-mince &#39;las muestras tumorales. Coloque el tubo de tejido completamente picada de nuevo en hielo, y repita con todos los tubos de muestras adicionales. </li><li> Transferencia de todas las muestras tumorales fragmentados en un único tubo cónico de 15 ml. Una pipeta P1000 con una punta estéril cortar / modificado funciona bien para este paso. Utilice adicional HBSS + / + para lavar / enjuagar los tubos ml de disociación 2 para asegurarse de que todas las muestras de tumor que se ha recogido en el tubo de 15 ml. Centrifugar la mezcla en centrifugar a 23 ° C, a 73 × g durante 5 min. </li><li> Aspirar fuera HBSS + / +, dejando el sedimento celular. Vuelva a suspender los fragmentos en una mezcla 1:1 de tripsina al 0,25%: 0,2% de colagenasa (disuelto en HBSS-/ -) - añadir lo suficiente para mantener los fragmentos tumorales en estrecha dospension. Vuelva a colocar el tornillo de la parte superior, y el lugar en el 37 ° C incubadora durante 30 min. Agite la mezcla de células al menos una vez durante la incubación para mantener los fragmentos en suspensión. Coloque el medio de cultivo Schwannoma en la incubadora para que se caliente en este momento también. </li><li> Después de la incubación, añadir 100 ml de FBS a cada tubo para inactivar la tripsina, y luego la muestra se centrifuga a 23 ° C, 73 xg durante 5 min. Con una pipeta, aspirar con cuidado tanto sobrenadante como sea posible sin molestar sedimento celular. Añadir los medios de cultivo calentado (N2/insulin/5% de FBS) a los fragmentos de tumor a una relación final de 01:02 fragmentos de tumor: medios de cultivo (por ejemplo, si hay 1 ml de fragmentos de tumor, añadir en 2 ml de calentado los medios de comunicación). </li><li> Prepararse para la trituración del tumor cortando la punta fuera de una punta de pipeta P1000 a un diámetro de aproximadamente 1-2 mm. Poco a poco se tritura la suspensión celular utilizando puntas de pipeta de diámetro cada vez más pequeños y más pequeños, hasta que pueda pipetear fácilmente la solución a través de un unaltpunta de la pipeta Ered P1000. Desplácese inmediatamente a una punta más pequeña una vez que usted puede pipetear fácilmente la solución del tumor a través de la punta - no más de triturar la muestra. Si una sola pieza más grande de bloques de tejido aspiración durante la trituración, tocando la punta de la pipeta en la parte inferior del tubo cónico a menudo permite la trituración continua. </li></ol> 5. Celular Plating <ol><li> Un 0,5 mm 3 fragmento de tejido es suficiente para sembrar 1 x 100 mm de plato, o 4 x diapositivas 8-well/4-well. Justo antes de la adición de la mezcla de medios de comunicación / celular, eliminar la solución de laminina de las placas de cultivo, pero no deje que las placas secas. </li><li> Para cada placa de 100 mm, disolver el fragmento de tumor en 10 ml de medios de cultivo calentado. </li><li> Para cada pocillo de un portaobjetos de 4-bien, utilizar 750 l de medios de cultivo calentado (3 ml para toda la diapositiva). </li><li> Para cada pocillo de un porta-8, así, utilizar 400 l de medios de cultivo calentado (3,2 ml para toda la diapositiva). </li><li> Los cultivos se incuban en 37 &amp;# 160; ° C, humedad del 100%, 6% de CO 2. Deje las culturas solo durante por lo menos 36 horas, y luego cambiar los medios de comunicación si la solución está amarilleado (ácido). De lo contrario, cambiar los medios de comunicación a las 72 horas, y después cada 2-3 días o cuando los medios de comunicación se convierten en ácido. Si los medios de comunicación se convierte en ácido al día durante varios días seguidos, esto es generalmente un indicio de contaminación bacteriana o de levadura potencial. </li></ol>

<ol>
	<li>Arthurs, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+treatment+modalities+for+vestibular+schwannoma.">A review of treatment modalities for vestibular schwannoma.</a> Neurosurg Rev. 34 (3), 265-277 (2011).</li><li>Evans, G. R., Lloyd, S. K., Ramsden, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+type+2.">Neurofibromatosis type 2.</a> Adv Otorhinolaryngol. 70, 91-98 (2001).</li><li>Fong, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+biology+and+novel+treatments+of+vestibular+schwannomas.">The molecular biology and novel treatments of vestibular schwannomas.</a> J Neurosurg. 115 (5), 906-914 (2011).</li><li>Jacob, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preclinical+validation+of+AR42,+a+novel+histone+deacetylase+inhibitor,+as+treatment+for+vestibular+schwannomas.">Preclinical validation of AR42, a novel histone deacetylase inhibitor, as treatment for vestibular schwannomas.</a> Laryngoscope. 122 (1), 174-189 (2012).</li><li>Evans, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neurofibromatosis+2+[Bilateral+acoustic+neurofibromatosis,+central+neurofibromatosis.">Neurofibromatosis 2 [Bilateral acoustic neurofibromatosis, central neurofibromatosis.</a> NF2, neurofibromatosis type II]. Genet Med. 11 (9), 599-610 (2009).</li><li>Anniko, M., Noren, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Human+Acoustic+Neurinoma+in+Organ-Culture+.1.+Methodological+Aspects.">The Human Acoustic Neurinoma in Organ-Culture .1. Methodological Aspects.</a> Acta Oto-Laryngologica. 91 (1-2), 47-53 (1981).</li><li>Manent, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+cell+sorting+for+enriching+Schwann+cells+from+adult+mouse+peripheral+nerves.">Magnetic cell sorting for enriching Schwann cells from adult mouse peripheral nerves.</a> J Neurosci Methods. 123 (2), 167-173 (2003).</li><li>Nair, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+cultures+of+human+vestibular+schwannoma:+selective+growth+of+schwannoma+cells.">Primary cultures of human vestibular schwannoma: selective growth of schwannoma cells.</a> Otol Neurotol. 28 (2), 258-263 (2007).</li><li>Baur, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laminin+promotes+differentiation,+adhesion+and+proliferation+of+cell+cultures+derived+from+human+acoustic+nerve+schwannoma.">Laminin promotes differentiation, adhesion and proliferation of cell cultures derived from human acoustic nerve schwannoma.</a> Acta Otolaryngol. 115 (4), 517-521 (1995).</li><li>Cravioto, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experimental+neurinoma+in+tissue+culture.">Experimental neurinoma in tissue culture.</a> Acta Neuropathol. 21 (2), 154-164 (1972).</li><li>Kaasinen, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Culturing+of+acoustic+neuroma--methodological+aspects.">Culturing of acoustic neuroma--methodological aspects.</a> Acta Otolaryngol Suppl. 520 Pt 1, 25-26 (1995).</li><li>Kredel, F. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tissue+Culture+of+Intracranial+Tumors+with+a+Note+on+the+Meningiomas.">Tissue Culture of Intracranial Tumors with a Note on the Meningiomas.</a> Am J Pathol. 4 (4), 337-340 (1928).</li><li>Murray, M. R., Stout, A. P., Bradley, C. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Schwann+cell+versus+fibroblast+as+the+origin+of+the+specific+nerve+sheath+tumor:+Observations+upon+normal+nerve+sheaths+and+neurilemomas+in+vitro.">Schwann cell versus fibroblast as the origin of the specific nerve sheath tumor: Observations upon normal nerve sheaths and neurilemomas in vitro.</a> Am J Pathol. 16 (1), 41-60 (1940).</li><li>Cravioto, H., Lockwood, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+behavior+of+acoustic+neuroma+in+tissue+culture.">The behavior of acoustic neuroma in tissue culture.</a> Acta Neuropathol. 12 (2), 141-157 (1969).</li><li>Lee, J. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+epigenetic+alterations+of+the+NF2+gene+in+sporadic+vestibular+schwannomas.">Genetic and epigenetic alterations of the NF2 gene in sporadic vestibular schwannomas.</a> PLoS One. 7 (1), 30418 (2012).</li><li>Wippold, F. J., 2nd, , et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuropathology+for+the+neuroradiologist:+Antoni+A+and+Antoni+B+tissue+patterns.">Neuropathology for the neuroradiologist: Antoni A and Antoni B tissue patterns.</a> AJNR Am J Neuroradiol. 28 (9), 1633-1638 (2007).</li><li>Mennel, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-term+tissue+culture+observations+of+experimental+primary+and+transplanted+nervous+system+tumors.">Short-term tissue culture observations of experimental primary and transplanted nervous system tumors.</a> J Neuropathol Exp Neurol. 39 (6), 639-660 (1980).</li><li>Rosenbaum, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+and+characterization+of+Schwann+cells+from+neurofibromatosis+type+2+patients.">Isolation and characterization of Schwann cells from neurofibromatosis type 2 patients.</a> Neurobiol Dis. 5 (1), 55-64 (1998).</li></ol>

No hay conflictos de interés a revelar.

Historia de cultivos in vitro Schwannoma Los esfuerzos por establecer en cultivos in vitro schwannoma comenzaron tan sólo unas décadas después de que el neuroma acústico inicial resección quirúrgica abierta se produjo en 1894 12. Los primeros cultivos fueron grabadas por Kredel en la década de 1920, que intentó sin éxito para crecer tumor triturado por &quot;método de la gota colgante&quot; 12 . Más tarde, los Dres....

Schwannomas vestibulares (VSS) representan Schwann células (SC), los tumores del nervio vestibular, lo que compromete el 10% de todas las neoplasias intracraneales 1-3. VS ocurre en (neurofibromatosis tipo 2, NF2) formas ya sea esporádica o familiar, tanto asociados a defectos de inactivación del gen supresor tumoral NF2 gen. El tratamiento para la VS es generalmente la resección quirúrgica o radiocirugía, sin embargo, la morbilidad de estos procedimientos incluyen sordera, neuropatías faciales, fuga de líquido cefalorraquídeo, el desequilibrio, y el nuevo crecimiento del tumor 1. Además no todos los pacientes son candidatos para la cirugía o la radiación aceptables. Tal morbilidad significativa, así como una falta de terapias alternativas ha impulsado la investigación en la biología molecular única de VS en la esperanza de desarrollar nuevos tratamientos de 3,4. El cultivo celular permite el análisis rápido, fácil, y en profundidad del comportamiento molecular y celular y la detección del potencial terapéutico comlibra. Históricamente, la falta de acceso a los especímenes de tejido fresco y el hecho de que las células no se inmortalizan schwannoma han obstaculizado significativamente el uso de cultivos primarios para la investigación de la tumorigénesis schwannoma. Para superar la limitada oferta de los cultivos primarios, la línea celular inmortalizada HEI193 VS se generó por transducción con VPH E6 y E7 oncogenes 5. Esta transducción oncogénico introdujo alteraciones moleculares y fenotípicos significativos a las células, que limitan su uso como un modelo para los tumores schwannoma humanos. Culturas SC derivados de líneas de ratones transgénicos que carecen de un gen NF2 funcional representan otra alternativa para investigar la tumorigénesis SC NF2 in vitro dependiente. Estas culturas, sin embargo no pueden recapitular la naturaleza heterogénea de las VS humana o dar cuenta de la conducta humana específica. La mayoría de las técnicas anteriores cultura VS requieren tiempos de procesamiento relativamente largas y complicadas técnicas de cultivo selectivos 6-8.Aquí se presenta un protocolo simple, reproducible para primaria VS cultivo celular con un proceso completo en menos de 3 horas, con un 95% de pureza de las células tumorales como se determina por inmunotinción.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly-L-Ornithine, 0.01% Solution</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">P4957</td>
			<td style="width:167px;">Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Laminin Mouse Protein&#160;</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">23017-015 / L2020</td>
			<td>Cell Culture Surface Treatment</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">0.2% Collagenase (dissolved in HBSS -/-)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">C2674</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.25% Trypsin</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">25200-056</td>
			<td>Dissociation Reagent</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TPS 100mm Round Culture Dish</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP93100</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 4 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256521</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Permanox 8 well slide</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">1256522</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Suction Filter Flask</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP99500</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">15ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91015</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">50ml Conical Tube</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TP91050</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2mL Round Bottom Tube</td>
			<td style="width:71px;">USA Scientific</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Scissor</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">14058-11</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Small Forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11251-20</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scalpel Handle</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">10004-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">#11 Scalpel Blade</td>
			<td style="width:71px;">Roboz</td>
			<td style="width:119px;">RS-9801-11</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Non-Tissue Culture 100mm Round Petri Dish</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-820-904</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">P1000 Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">P3963-1000</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Serological Pipetteman</td>
			<td style="width:71px;">Bioexpress</td>
			<td style="width:119px;">R3073-2P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sterile, Non-Filtered P1000 Pipette Tips</td>
			<td style="width:71px;">Midwest Scientific</td>
			<td style="width:119px;">TD1250R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Insulated Ice Cooler</td>
			<td style="width:71px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Culture Hood&#160;</td>
			<td style="width:71px;">Baker</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Centrifuge&#160;</td>
			<td style="width:71px;">Eppendorf -5810R</td>
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			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) +/+ (w/ Ca2+, Mg2+)&#160;</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="42" style="height:42px;width:216px;">Hanks Balanced Salt Solution (HBSS) -/- (w/out Ca2+, Mg2+)</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="63" style="height:63px;width:216px;">Dulbecco&#8217;s Modified Eagle Medium (DMEM), High Glucose, w/ Phenol Red</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="21" style="height:21px;width:216px;">N2 Supplement</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="21" style="height:21px;width:216px;">Fetal Bovine Serum</td>
			<td style="width:71px;">Gibco</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td>Schwannoma Culture and Media Components</td>
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			<td height="21" style="height:21px;width:216px;">Bovine Insulin (1mg/ml 200x)</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">I6634</td>
			<td>Schwannoma Culture and Media Components</td>
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primary culture of human vestibular schwannomas

Schwannomas vestibulares (VSS) representan Schwann células (SC), los tumores del nervio vestibular, lo que compromete el 10% de todas las neoplasias intracraneales. VS ocurre en (neurofibromatosis tipo 2, NF2) formas ya sea esporádica o familiar, tanto asociados a defectos de inactivación del gen supresor tumoral NF2 gen. El tratamiento para la VS es generalmente la resección quirúrgica o radiocirugía, sin embargo, la morbilidad de estos procedimientos ha conducido investigaciones sobre los tratamientos menos invasivos. Históricamente, la falta de acceso a los especímenes de tejido fresco y el hecho de que las células no se inmortalizan schwannoma han obstaculizado significativamente el uso de cultivos primarios para la investigación de la tumorigénesis schwannoma. Para superar la limitada oferta de los cultivos primarios, la línea celular inmortalizada HEI193 VS se generó por transducción con VPH E6 y E7 oncogenes. Esta transducción oncogénico introdujo alteraciones moleculares y fenotípicos significativos a las células, que limitan su uso como un modelo para s humanostumores chwannoma. Por lo tanto, ilustramos un protocolo reproducible simplificado para la cultura de los derechos humanos primaria células VS. Esta técnica permite dominar fácilmente para investigaciones moleculares y celulares que recapitulan con más precisión la complejidad de la enfermedad de VS.

La fragmentación del tumor correcta es esencial para la siembra óptima y el crecimiento en las placas de cultivo de tejido (Figura 1). Identificación de células schwannoma durante los primeros días después de la siembra es a menudo difícil, debido a oscurecer cantidades de residuos celulares y las células rojas de la sangre. Por el 4 º o 5 º día, las extensiones celulares cerca de fragmentos tumorales adherentes crearán reconocible &#39;cordón&#39; patrones entre los e...

Watch this Scientific Journal Video about Primary Culture of Human Vestibular Schwannomas at JoVE.com

Primary Culture of Human Vestibular Schwannomas

Vestibular schwannomas (VS) son tumores no malignos de células (SC) origen Schwann, asociadas a mutaciones en el gen supresor de tumores NF2. Se presenta un protocolo reproducible, eficiente para la enseñanza primaria humanos VS cultivo celular que permite la manipulación experimental molecular y celular y el análisis y recapitula la naturaleza heterogénea de la enfermedad humana.

Cultivo primario de Human vestibular schwannomas

Vestibular schwannomas (VSs) represent Schwann cell (SC) tumors of the vestibular nerve, compromising 10% of all intracranial neoplasms. VSs occur in ...

primary-culture-of-human-vestibular-schwannomas

Research

JoVE Journal

Medicine

12.7K vistas.  University of Iowa Hospitals and Clinics.  Vestibular schwannomas (VS) son tumores no malignos de células (SC) origen Schwann, asociadas a mutaciones en el gen supresor de tumores NF2. Se presenta un protocolo reproducible, eficiente para la enseñanza primaria humanos VS cultivo celular que permite la manipulación experimental molecular y celular y el análisis y recapitula la naturaleza heterogénea de la enfermedad humana. 

Video: Primary Culture of Human Vestibular Schwannomas

Skin Punch Biopsy Explant Culture for Derivation of Primary Human Fibroblasts

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts in this protocol have been successfully used in the derivation of induced pluripotent stem cells to model disease1. Early passages of these fibroblasts can also be used for cell-based functional assays to study specific disease pathways, mechanisms2 and subsequent drug screening approaches. The advantage of the presented protocol over enzymatic procedures are 1) the reproducibility of the technique from small amounts of tissue derived from older patients, e.g. patients affected with Parkinson's disease, 2) the technically simple approach over more challenging methodologies using enzymatic treatments, and 3) the time consideration: this protocol takes 15-20 min and can be performed immediately after biopsy arrival. Enzymatic treatments can take up to 4 hr and have the problems of overdigestion, reduction of cell viability and subsequent attachment of cells when not handled properly. This protocol describes the dissection and preparation of a 4-mm human skin biopsy for derivation of a fibroblast culture and has a very high success rate which is important when dealing with patient-derived tissue samples. In this culture, keratinocytes migrate out of the biopsy tissue within the first week after preparation. Fibroblasts appear 7-10 days after the first outgrowth of keratinocytes. DMEM high glucose media supplemented with 20% FBS favors the growth of fibroblasts over keratinocytes and fibroblasts will overgrow the keratinocytes. After 2 passages keratinocytes have been diluted out resulting in relatively homogenous fibroblast cultures which expresses the fibroblast marker SERPINH1 (HSP-47). Using this approach, 15-20 million fibroblasts can be derived in 4-8 weeks for cell banking. The skin dissection takes about 15-20 min, cells are then monitored once a day under the microscope, and media is changed every 2-3 days after attachment and outgrowth of cells.

The fibroblast explant culture protocol from human skin punch biopsies is a technically robust and simple way to derive skin cells within 4-8 weeks for banking of about 15-20 million cells at a low passage number.

Piel biopsia en sacabocados explante cultura para Derivación de fibroblastos humanos primarios

Tissues and cell lines derived from an individual with disease are ideal sources to study disease-related cellular phenotypes. Patient-derived fibroblasts ...

Investigación

Medicina

In vitro Organoid Culture of Primary Mouse Colon Tumors

Several human and murine colon cancer cell lines have been established, physiologic integrity of colon tumors such as multiple cell layers, basal-apical polarity, ability to differentiate, and anoikis are not maintained in colon cancer derived cell lines. The present study demonstrates a method for culturing primary mouse colon tumor organoids adapted from Sato T et al. 1, which retains important physiologic features of colon tumors. This method consists of mouse colon tumor tissue collection, adjacent normal colon epithelium dissociation, colon tumor cells digestion into single cells, embedding colon tumor cells into matrigel, and selective culture based on the principle that tumor cells maintain growth on limiting nutrient conditions compared to normal epithelial cells.
The primary tumor organoids if isolated from genetically modified mice provide a very useful system to assess tumor autonomous function of specific genes. Moreover, the tumor organoids are amenable to genetic manipulation by virus meditated gene delivery; therefore signaling pathways involved in the colon tumorigenesis could also be extensively investigated by overexpression or knockdown. Primary tumor organoids culture provides a physiologic relevant and feasible means to study the mechanisms and therapeutic modalities for colon tumorigenesis.

In vitro Organoid Culture of Primary Mouse Colon Tumors

A simple method to establish primary murine colon tumor organoid is described. This method utilizes the feature that colon tumor cells survive and grow into organoids in media containing limited growth factors, whereas normal colon epithelial do not.

In vitro organoide Cultura de tumores de colon primarios Ratón

Several  human and murine colon cancer cell lines have been established, physiologic  integrity of colon tumors such as multiple cell layers, basal-apical ...

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 ...

A Three-dimensional Tissue Culture Model to Study Primary Human Bone Marrow and its Malignancies

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods rely on the ability of cells either to attach to a solid substratum of a tissue culture dish or to grow in suspension in liquid medium. Multiple immortal cell lines have been created and grown using such approaches, however, these methods frequently fail when primary cells need to be grown ex vivo. Such failure has been attributed to the absence of the appropriate extracellular matrix components of the tissue microenvironment from the standard systems where tissue culture plastic is used as a surface for cell growth. Extracellular matrix is an integral component of the tissue microenvironment and its presence is crucial for the maintenance of physiological functions such as cell polarization, survival, and proliferation. Here we present a 3-dimensional tissue culture method where primary bone marrow cells are grown in extracellular matrix formulated to recapitulate the microenvironment of the human bone (rBM system). Embedded in the extracellular matrix, cells are supplied with nutrients through the medium supplemented with human plasma, thus providing a comprehensive system where cell survival and proliferation can be sustained for up to 30 days while maintaining the cellular composition of the primary tissue. Using the rBM system we have successfully grown primary bone marrow cells from normal donors and patients with amyloidosis, and various hematological malignancies. The rBM system allows for direct, in-matrix real time visualization of the cell behavior and evaluation of preclinical efficacy of novel therapeutics. Moreover, cells can be isolated from the rBM and subsequently used for in vivo transplantation, cell sorting, flow cytometry, and nucleic acid and protein analysis. Taken together, the rBM method provides a reliable system for the growth of primary bone marrow cells under physiological conditions.

In standard culture methods cells are taken out of their physiological environment and grown on the plastic surface of a dish. To study the behavior of primary human bone marrow cells we created a 3-D culture system where cells are grown under conditions recapitulating the native microenvironment of the tissue.

Un tejido tridimensional Cultura modelo para estudiar primaria médula ósea humana y sus malignidades

Tissue culture has been an invaluable tool to study many aspects of cell function, from normal development to disease. Conventional cell culture methods ...

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 ...

Isolation of Primary Human Decidual Cells from the Fetal Membranes of Term Placentae

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized stromal cells and immune cells, are responsible for the secretion of hormonal and inflammatory factors which are critical for successful blastocyst implantation, placental development and play a role in the initiation of labor at term and preterm. Many pregnancy complications can arise from perturbations of a fine balance of different cell populations comprising decidua. Alterations in the proportion of specific decidual cell types may disrupt these crucial processes and increase the risk of developing serious complications of pregnancy, such as embryo implantation failure, intrauterine growth restriction, preeclampsia and preterm labor. The protocol outlined here demonstrates a cost and time effective method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae. By combining enzymatic digestion and gentle mechanical disruption of the decidual tissue, a high yield of decidual cells was obtained with virtually no chorion contamination. Importantly, isolated decidual cells were characterized (stromal cells (55-60%), leukocytes (35%), epithelial (1%) or trophoblast (0.01%) cells) and maintained high viability (80%) which was confirmed by multicolor imaging flow cytometry assay. This protocol is specific to the decidua parietalis and can be adapted to first and second trimester placentae. Once isolated, decidual cells can be used for a multitude of experimental applications aiming to understand the role of different decidual cell sub-populations in pregnancy complications.

This protocol demonstrates a method for the isolation of primary human decidual cells collected from the fetal membranes of term placentae which can be used for a variety of applications (i.e. immunocytochemistry, flow cytometry, etc.) aiming to study the role of different cell populations in pregnancy complications.

Aislamiento de células Decidual humanas primarias de las membranas fetales de la placenta de término

The decidua, also known as the pregnant endometrium, is a critically important reproductive tissue. Decidual cells, comprised mainly of decidualized ...

Muscle Imbalances: Testing and Training Functional Eccentric Hamstring Strength in Athletic Populations

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite of these eccentric hamstring actions are concentric quadriceps actions, where the larger and likely stronger quadriceps straighten the knee. Therefore, to stabilize the lower limbs during movement, the hamstrings must eccentrically combat against the strong knee-straightening torque of the quadriceps. As such, eccentric hamstring strength expressed relative to concentric quadricep strength is commonly referred to as the &#34;functional ratio&#34; as most movements in sports require simultaneous concentric knee extension and eccentric knee flexion. To increase the strength, resiliency, and functional performance of the hamstrings, it is necessary to test and train the hamstrings at different eccentric speeds. The main purpose of this work is to provide instructions for measuring and interpreting eccentric hamstring strength. Techniques for measuring the functional ratio using isokinetic dynamometry are provided and sample data will be compared. Additionally, we briefly describe how to address hamstring strength deficiencies or unilateral strength differences using exercises that specifically focus on increasing eccentric hamstring strength.

The hamstrings are a group of muscles that are sometimes problematic for athletes, resulting in soft tissue injury in the lower limbs. To prevent such injuries, functional training of the hamstrings requires intensive eccentric contractions. Additionally, hamstring function should be tested in relation to quadricep function at different contraction speeds.

Los desequilibrios musculares: Pruebas y entrenamiento de fuerza funcional excéntrico isquiotibiales en poblaciones deportivas

Many hamstring injuries that occur during physical activity occur while the muscles are lengthening, during eccentric hamstring muscle actions. Opposite ...

Surgical Labyrinthectomy of the Rat to Study the Vestibular System

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including surgical or chemical labyrinthectomy and vestibular neurectomy. Surgical labyrinthectomy is a relatively simple, reliable, and rapid method. Here, we describe the surgical technique for rat labyrinthectomy. A postauricular incision is made under general anesthesia to expose the external auditory canal and the tympanic membrane, after which the tympanic membrane and the ossicles are removed without the stapes. The stapes artery, which is located between the stapes and the oval window, is a vulnerable structure and must be preserved to obtain a clear surgical field. A hole to fenestrate the vestibule is made with a 2.1-mm drill bur superior to the stapes. Then, 100% ethanol is injected through this hole and aspirated several times. Meticulous dissection under a microscope and careful bleeding control are essential to obtain reliable results. Symptoms of vestibular loss, such as nystagmus, head tilting, and a rolling motion, are seen immediately after surgery. The rotarod or rotation chair test can be used to objectively and quantitatively evaluate the vestibular function.

This protocol describes the surgical labyrinthectomy of a rat, which is a useful method for studying the vestibular system.

Labyrinthectomy quirúrgica de la rata para el estudio del sistema Vestibular

To study the vestibular system or the vestibular compensation process, a number of methods have been developed to cause vestibular damage, including ...

Characterization of a Novel Human Organotypic Retinal Culture Technique

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal pigment epithelium-choroid (RPE-choroid) and sclera, the integrity of the fragile retina can easily be compromised. The aim of this study was to develop a novel HORC model that contains the retina, RPE-choroid and sclera to maintain retinal integrity when culturing retinal explants.
After cutting circumferentially along the limbus to remove iris and lens, four deep incisions were made to flatten the eyecup. In contrast to previous HORC protocols, a trephine was used to cut through not only the retina but also the RPE-choroid and sclera. The resultant triple-layered explants were cultured for 72 h. Hematoxylin and Eosin staining (H&#38;E) was used to assess anatomical structures and retinal explants were further characterized by immunohistochemistry (IHC) for apoptosis, M&#252;ller cell integrity and retinal inflammation. To confirm the possibility of disease induction, explants were exposed to high glucose (HG) and pro-inflammatory cytokines (Cyt), to mimic diabetic retinopathy (DR). The Luminex magnetic bead assay was used to measure DR-related cytokines released into the culture medium.
H&#38;E staining revealed distinct retinal lamellae and compact nuclei in retinal explants with the underlying RPE-choroid and sclera, while retinas without the underlying structures exhibited reduced thickness and severe nuclei loss. IHC results indicated absence of apoptosis and retinal inflammation as well as preserved M&#252;ller cell integrity. The Luminex&#160;assays showed significantly increased secretion of DR-associated pro-inflammatory cytokines in retinal explants exposed to HG + Cyt relative to baseline levels at 24 h.
We successfully developed and characterized a novel HORC protocol in which retinal integrity was preserved without apoptosis or retinal inflammation. Moreover, the induced secretion of DR-associated pro-inflammatory biomarkers when exposing retinal explants to HG + Cyt suggests that this model could be used for clinically translatable retinal disease studies.

This study aims to develop a novel human organotypic retinal culture (HORC) model that prevents compromising retinal integrity during explant handling. This is achieved by culturing the retina with the overlying vitreous and the underlying retinal pigment&#160;epithelium-choroid (RPE-choroid) and sclera.

Caracterización de una nueva técnica de cultivo de retina organotípica humana

Previous human organotypic retinal culture (HORC) models have utilized detached retinas; however, without the structural support conferred by retinal ...