This work was supported by the NRF grant HUJ-CREATE - Cellular and Molecular Mechanisms of Inflammation.

Los animales fueron alojados en condiciones libres de patógenos en el cumplimiento de las directrices institucionales hasta la eutanasia (El Cuidado de Animales institucional y el empleo Comisión (IACUC) directrices de la Universidad Nacional de Singapur y el Comité Nacional Asesor para el Laboratorio de Investigación Animal (NACLAR) directrices). 1. Los ratones <ol><li> Ordene un ratón del fondo genético apropiado. Este protocolo se basa en el tejido derivado de un ratón C57BL / 6. </li></ol> 2. Preparación de medio completo <ol><li> Preparar medio completo mediante la adición de los siguientes componentes a Roswell Park Memorial Institute (RPMI) 1640: 10% de suero de ternera fetal (FCS), 50 mM 2-mercaptoetanol, 100 mM de asparagina, glutamina 2 mM, solución de penicilina-estreptomicina al 1%. </li></ol> 3. Preparación de soluciones de enzima <ol><li> Prepare la solución de colagenasa D en un tubo de fondo cónico de 15 ml. <ol><li> Pesar 10 mg de colagenasa D. Dissolve colagenasa D en 4 ml de medio completo. </li></ol></li><li> Prepare la solución de pronasa en un tubo de 1,7 ml de microcentrífuga. <ol><li> Pesar 10 mg de pronasa. </li><li> Añadir 5 l de tampón Tris 1 M (pH 8,0). Añadir 1 l de 0,5 M EDTA (pH 8,0). </li><li> Rellene con 494 l de agua estéril. </li><li> Incubar la solución de pronasa a 37 ° C en un baño de agua durante 30 minutos. </li></ol></li></ol> 4. Preparación de colagenasa D-pronasa Mix (≤2 colas) Nota: Realice los pasos siguientes en una campana de cultivo celular estéril. <ol><li> Añadir 250 l de solución de pronasa a 4 ml de solución de colagenasa D. </li><li> Pasar la colagenasa-D pronasa mezcla a través de un filtro de jeringa de 0,2 micras en un tubo de fondo cónico de 15 ml estéril. </li></ol> 5. Extracción de fibroblastos de oreja y rabo tejidos <ol><li> La eutanasia a los ratones de acuerdo con las directrices institucionales apropiados.</li><li> Coloque autoclave instrumentos quirúrgicos (tijeras y pinzas) en la campana de cultivo celular. </li><li> Añadir 10 ml de medio completo en dos platos de 10 cm de cultivo de células cada uno. </li><li> Cortar las orejas (~ 1 cm de radio) y 5 cm de la cola (de la punta de la cola) de un ratón con tijeras y se incuba durante 5 min en etanol 40 ml 70% en un tubo de fondo cónico de 50 ml estéril. </li><li> Oídos Aire seco y cola colocándolos en una abierta 10 cm placa de cultivo celular en la campana durante 5 min. Una vez secas, oído transferencia y piezas de la cola a dos placas de cultivo etiquetados &quot;orejas&quot; y &quot;cola&quot; que contiene 10 ml de medio completo como se describe en el paso 5.3. </li><li> Quitar el pelo de las orejas y la cola con unas tijeras. </li><li> Cortar orejas y rabo en trozos más pequeños de 3 mm de tamaño con unas tijeras. </li><li> Transferir los tejidos cortados en 1,8 ml viales criotubo etiquetados &quot;orejas&quot; y &quot;cola&quot; y añadir la solución D-pronasa suficiente de colagenasa para el volumen para alcanzar la marca de 1,8 ml en el frasco. </li><li> Pata los viales criotubo horizontalmente en una coctelera y agitar las muestras a 200 rpm durante 90 minutos a 37 ° C. </li><li> Después de 90 minutos de incubación, retire los viales criotubo de la coctelera y colocar los viales en el capó. </li><li> Añadir 10 ml de medio completo en dos 10 cm placas de cultivo de células, etiquetados &quot;oídos&quot; y &quot;cola&quot; cada uno, y poner un filtro de 70 micras de células en cada plato. </li><li> Coloca los tejidos del oído y de la cola digeridos en el colador de 70 micras de células en los platos en consecuencia etiquetados preparados en el paso 5.11 y con fuerza moler los tejidos utilizando una jeringa de émbolo 10 ml durante&gt; 5 min. Agite el filtro de células de vez en cuando en el medio para lavar las células del filtro de células. </li><li> Pipetear la suspensión de células de cada plato en tubos de fondo dos 15 ml cónicos etiquetados &quot;orejas&quot; y &quot;cola&quot;. Lave el plato y el colador con otros 10 ml de medio completo y añadir el medio a los tubos de fondo cónico de 15 ml apropiadas. </li><li> Centrifugar la célulasuspensión durante 7 min a 580 xg y ~ 4 ° C utilizando una centrífuga refrigerada celular. </li><li> Aspirar el sobrenadante, añadir 10 ml de medio completo al sedimento celular en los 15 ml de tubo de fondo cónico y resuspender las células. </li><li> Repita el paso 5.14 y 5.15. </li></ol> 6. Cultivo de mezcla de células <ol><li> Eliminar el sobrenadante. Asegúrese de que el sedimento celular se mantiene inalterado. </li><li> Vuelva a suspender las células en 10 ml de medio completo y añadir la mezcla correspondiente a dos placas de cultivo celular de 10 cm etiquetados &quot;orejas&quot; y &quot;cola&quot;. </li><li> Añadir 10 l solución de anfotericina B (solución madre: 250 mg / ml) a la cultura. </li><li> Se incuban las células a 37 ° C en un humidificado 5% de CO 2 incubadora. </li><li> En el tercer día, sustituir el medio con medio completo fresco que contiene 10 ml 10 l de anfotericina B para eliminar los residuos (Figuras 1 y 2). </li></ol> 7. Sub-cultivo de fibroblastos <ol><li> When cultivo alcanza alrededor de 70% de confluencia (día 3-4 de la cultura) se elimina el medio y se lavan las células con solución salina estéril fosfato 5 ml 1x tamponada (PBS). </li><li> Retirar PBS y añadir 2 ml de solución de tripsina-EDTA 1x estéril a las células. </li><li> Se incuban las células durante 5 min a 37 ° C en un humidificado 5% de CO 2 incubadora. </li><li> Después de 5 minutos, toque suavemente la placa de cultivo y añadir 6 ml de medio completo a las células. </li><li> Transferir la suspensión celular a un tubo de 15 ml de fondo cónico y girar el tubo durante 5 min a 450 xg y ~ 4 ° C utilizando una centrífuga refrigerada celular. </li><li> Eliminar el sobrenadante y volver a suspender suavemente el sedimento de células en 5 ml de medio completo. </li><li> Semilla 2 x 10 5 células en una placa de 10 cm de cultivo celular y se incuban las células a 37 ° C en un humidificado 5% de CO 2 incubadora durante 3-4 días antes de repetir los pasos 7.1 a 7.6. </li></ol> 8. Preparación de Orejas para Embarque Nota: Realice tque los pasos subsiguientes en una campana de cultivo celular estéril. <ol><li> La eutanasia a los ratones de acuerdo con las directrices institucionales apropiados. </li><li> Coloque las tijeras y fórceps autoclave a la campana de cultivo celular. </li><li> Añadir 50 ml de medio completo en un tubo de fondo cónico de 50 ml. </li><li> Cortar orejas (~ 1 cm de radio) de un ratón con tijeras. </li><li> La transferencia de los oídos en el tubo de fondo cónico de 50 ml (como se ha preparado en el paso 3) el uso de fórceps. </li><li> Sellar el tubo inferior 50 ml cónico con parafina antes de enviar a temperatura ambiente en una caja apropiada. </li></ol>

<ol>
	<li>Fitzpatrick, L. E., McDevitt, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-derived+matrices+for+tissue+engineering+and+regenerative+medicine+applications.">Cell-derived matrices for tissue engineering and regenerative medicine applications.</a> Biomater Sci. 3 (1), 12-24 (2015).</li><li>Elenbaas, 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=Human+breast+cancer+cells+generated+by+oncogenic+transformation+of+primary+epithelial+cells.">Human breast cancer cells generated by oncogenic transformation of primary epithelial cells.</a> Genes Dev. 15 (1), 50-65 (2001).</li><li>Stansley, B., Post, J., Hensley, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparative+review+of+cell+culture+systems+for+the+study+of+microglial+biology+in+Alzheimer's+disease.">A comparative review of cell culture systems for the study of microglial biology in Alzheimer's disease.</a> J Neuroinflammation. 9 (1), 115 (2012).</li><li>Lim, J., Dobson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+transfection+of+HUVEC+and+MEF+cells+using+DNA+complexes+with+magnetic+nanoparticles+in+an+oscillating+field.">Improved transfection of HUVEC and MEF cells using DNA complexes with magnetic nanoparticles in an oscillating field.</a> J Genet. 91 (2), 223-227 (2012).</li><li>Li, 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=High-efficiency+transduction+of+fibroblasts+and+mesenchymal+stem+cells+by+tyrosine-mutant+AAV2+vectors+for+their+potential+use+in+cellular+therapy.">High-efficiency transduction of fibroblasts and mesenchymal stem cells by tyrosine-mutant AAV2 vectors for their potential use in cellular therapy.</a> Hum Gene Ther. 21 (11), 1527-1543 (2010).</li><li>Patel, M., Yang, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+reprogramming+somatic+cells+to+induced+pluripotent+stem+cells.">Advances in reprogramming somatic cells to induced pluripotent stem cells.</a> Stem Cell Rev. 6 (3), 367-380 (2010).</li><li>Newman, A. C., Nakatsu, M. N., Chou, W., Gershon, P. D., Hughes, C. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+requirement+for+fibroblasts+in+angiogenesis:+fibroblast-derived+matrix+proteins+are+essential+for+endothelial+cell+luman+formation.">The requirement for fibroblasts in angiogenesis: fibroblast-derived matrix proteins are essential for endothelial cell luman formation.</a> Mol Biol Cell. 22 (20), 3791-3800 (2011).</li><li>Ohlund, D., Elyada, E., Tuveson, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fibroblast+heterogeneity+in+the+cancer+wound.">Fibroblast heterogeneity in the cancer wound.</a> J Exp Med. 211 (8), 1503-1523 (2014).</li><li>Guo, S., Dipietro, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factors+affecting+wound+healing.">Factors affecting wound healing.</a> J Dent Res. 89 (3), 219-229 (2010).</li><li>Werner, S., Krieg, T., Smola, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Keratinocyte-fibroblast+interactions+in+wound+healing.">Keratinocyte-fibroblast interactions in wound healing.</a> J Invest Dermatol. 127 (5), 998-1008 (2007).</li><li>Shen, Y. 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=Genome-derived+cytosolic+DNA+mediates+type+I+interferon-dependent+rejection+of+B+cell+lymphoma+cells.">Genome-derived cytosolic DNA mediates type I interferon-dependent rejection of B cell lymphoma cells.</a> Cell Rep. 11 (3), 460-473 (2015).</li><li>Seluanov, A., Vaidya, A., Gorbunova, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Establishing+primary+adult+fibroblast+cultures+from+rodents.">Establishing primary adult fibroblast cultures from rodents.</a> J Vis Exp. (44), (2010).</li><li>Baglole, C. 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=Isolation+and+phenotypic+characterization+of+lung+fibroblasts.">Isolation and phenotypic characterization of lung fibroblasts.</a> Methods Mol Med. 117, 115-127 (2005).</li><li>Alt, E., 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=Fibroblasts+share+mesenchymal+phenotypes+with+stem+cells,+but+lack+their+differentiation+and+colony-forming+potential.">Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential.</a> Biol Cell. 103 (4), 197-208 (2011).</li><li>Kuilman, T., Michaloglou, C., Mooi, W. J., Peeper, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+essence+of+senescence.">The essence of senescence.</a> Genes Dev. 24 (22), 2463-2479 (2010).</li><li>Lander, M. R., Moll, B., Rowe, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+procedure+for+culture+of+cells+from+mouse+tail+biopsies:+brief+communication.">A procedure for culture of cells from mouse tail biopsies: brief communication.</a> J Natl Cancer Inst. 60 (2), 477-478 (1978).</li><li>Moore, C. B., Allen, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+ear+fibroblast+derivation+from+mice.">Primary ear fibroblast derivation from mice.</a> Methods Mol Biol. (1031), 65-70 (2013).</li><li>Liu, 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=Generation+of+stable+pluripotent+stem+cells+from+NOD+mouse+tail-tip+fibroblasts.">Generation of stable pluripotent stem cells from NOD mouse tail-tip fibroblasts.</a> Diabetes. 60 (5), 1393-1398 (2011).</li></ol>

The authors declare no conflict of financial interests.

Aquí le ofrecemos un procedimiento experimental simple, barato y rápido para establecer cultivos de fibroblastos primarios de las orejas y la cola de los ratones. La extracción debe resultar en fibroblastos en división adherentes y rápidamente dentro de 3 días post-aislamiento del tejido. Una limitación importante de células primarias es la senescencia, una detención del crecimiento permanente 15. Utilizando el protocolo, cultivos de fibroblastos se pueden pasaron durante 5 a 6 veces antes de fibrobl...

Las células primarias se derivan de tejido y se cultivaron viven bajo condiciones in vitro. Se asume generalmente que las células primarias se asemejan más el estado fisiológico y el fondo genético del tejido del que se derivan de líneas celulares inmortalizadas o tumorales 1. Por esa razón, las células primarias representan un modelo útil para el estudio de cuestiones biológicas 2,3. Sin embargo, a diferencia de las líneas celulares establecidas que crecen indefinidamente, células primarias, finalmente, se someten a la senescencia en la cultura y necesitan ser restablecido con frecuencia. Células primarias comúnmente usados ​​incluyen fibroblastos, células epiteliales, células endoteliales, células T, células B, macrófagos derivados de médula ósea (BMDM) y células dendríticas derivadas de médula ósea (BMDC). Los fibroblastos se utilizan a menudo como modelo de cultivo celular primario. Ofrecen ventajas clave sobre otras células primarias. Los cultivos celulares son fáciles de establecer, fácilmente mantenido y no requieren Purificación de las células antes de la cultura. Tienen rápida proliferación inicial y sin necesidad de protocolos de medio o de activación especializados. Los fibroblastos se pueden transfectar eficientemente usando biológicos, químicos, físicos y protocolos de 4,5. Hay una posibilidad de almacenar oídos por hasta 10 días a temperatura ambiente antes de establecer cultivos celulares. Cultivos de fibroblastos son propicias para la visualización de los procesos citoplásmicos y adecuado para la reprogramación en células madre pluripotentes inducidas (iPS) 6. Los fibroblastos son células importantes del tejido conectivo que secretan las proteínas de colágeno y matriz extracelular 7. Ellos proporcionan el marco estructural en muchos tejidos 8 y juegan un papel esencial en la curación de heridas y reparación de tejidos 9,10. A continuación, describimos una (&lt;4 h) Protocolo simple y rápida para establecer cultivos de fibroblastos de las orejas y la cola de los ratones 11. El protocolo requiere un mínimo de ratónexperiencia para la cosecha de los tejidos (en contraste con otros protocolos 12,13) ​​y se puede utilizar para establecer cultivos de oídos almacenados en un medio a temperatura ambiente durante hasta 10 días.

<table border="0" cellpadding="0" cellspacing="0" width="642">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI-1640</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SH30027.01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Fetal Calf Serum</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30160.03</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">2-mercaptoethanol</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">M3148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Asparagine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A4159</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glutamine</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8540</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penicillin/Streptomycin</td>
			<td style="width:140px;">HyClone</td>
			<td style="width:119px;">SV30010</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">107017</td>
			<td>Absolute for analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase D</td>
			<td style="width:140px;">Roche Diagnostics</td>
			<td style="width:119px;">11088866001</td>
			<td>From Clostridium histolyticum, lyophilized, non-sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Pronase protease</td>
			<td style="width:140px;">Merck Millipore</td>
			<td style="width:119px;">53702</td>
			<td>From Streptomyces griseus&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris buffer (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">1415</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.5M EDTA (pH 8)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-1053</td>
			<td>Biotechnology grade</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">10X Phosphate Buffered Saline (PBS)</td>
			<td style="width:140px;">1st BASE</td>
			<td style="width:119px;">BUF-2040-10X4L</td>
			<td>Ultra pure grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin-EDTA solution 10X</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">59418C-100ML</td>
			<td>0.5% trypsin, 0.2% EDTA, trypsin gamma irradiated by SER-TAIN process, without phenol red, in saline</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Amphotericin B</td>
			<td style="width:140px;">Sigma-Aldrich</td>
			<td style="width:119px;">A2492-20ml</td>
			<td>250 &#956;g/ml in deionized water, sterile-filtered</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Scissors</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Forcep</td>
			<td style="width:140px;">Aesculap</td>
			<td style="width:119px;">AE-BD312R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">0.2 &#956;M syringe filter</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;">16534</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70 &#956;M cell strainer</td>
			<td style="width:140px;">SPL</td>
			<td style="width:119px;">93070</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Syringe plunger</td>
			<td style="width:140px;">Terumo</td>
			<td style="width:119px;">SS+10L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cryovial tube</td>
			<td style="width:140px;">NUNC</td>
			<td style="width:119px;">368362</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1.7 ml microcentrifuge tube</td>
			<td style="width:140px;">Axygen</td>
			<td style="width:119px;">MCT-175-C</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">10 cm cell culture dish</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">664160</td>
			<td>Cell culture treated dish&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">188271</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 ml conical bottom tube</td>
			<td style="width:140px;">Greiner</td>
			<td style="width:119px;">227261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Water bath</td>
			<td style="width:140px;">GFL</td>
			<td style="width:119px;">1002</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Centrifuge</td>
			<td style="width:140px;">Eppendorf</td>
			<td style="width:119px;">5810R</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Incubation shaker</td>
			<td style="width:140px;">Sartorius Stedim</td>
			<td style="width:119px;"></td>
			<td>Certomat-BS1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Zeiss Axiovert 25 light microscope</td>
			<td style="width:140px;">Carl Zeiss AG</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

generating primary fibroblast cultures from mouse ear and tail tissues

Las células primarias se derivan directamente de tejidos y se cree que son más representativa del estado fisiológico de las células in vivo que las líneas celulares establecidas. Sin embargo, cultivos de células primarias por lo general tienen una vida finita y deben ser restablecido con frecuencia. Los fibroblastos son una fuente fácilmente accesible de células primarias. En este caso, hablamos de un procedimiento experimental simple y rápida para establecer cultivos de fibroblastos primarios de las orejas y la cola de los ratones. El protocolo puede ser utilizado para establecer cultivos de fibroblastos primarios de los oídos almacenados a temperatura ambiente durante hasta 10 días. Cuando el protocolo se sigue cuidadosamente, las contaminaciones son poco probable que ocurra a pesar de la utilización de tejido no estéril almacenada por un tiempo prolongado en algunos casos. Los fibroblastos proliferan rápidamente en la cultura y se puede ampliar a un número considerable antes de someterse a la senescencia replicativa.

Extracción de fibroblastos de resultados de tejidos en una cantidad significativa de restos de tejido (Figura 1). En contraste con restos de tejido, los fibroblastos se adhieren a las superficies de plástico de cultivo de tejido entre el día 1 y 3 de la cultura. El medio de cultivos de fibroblastos se puede cambiar de forma segura en el día 3 de la cultura, lo que debería reducir significativamente los niveles de residuos presentes en la cultura (Figura 2). Los fibroblastos muestra...

Watch this Scientific Journal Video about Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues at JoVE.com

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Se describe un procedimiento experimental sencillo y rápido para la generación de fibroblastos primarios de las orejas y la cola de los ratones. El procedimiento no requiere una formación especial de los animales y se puede utilizar para la generación de cultivos de fibroblastos de los oídos almacenados a temperatura ambiente durante hasta 10 días.

Culturas Generación de fibroblastos primarios de mouse de oreja y rabo tejidos

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

generating-primary-fibroblast-cultures-from-mouse-ear-and-tail-tissues

Research

JoVE Journal

Developmental Biology

33.6K vistas.  Yong Loo Lin School of Medicine, National University of Singapore.  Se describe un procedimiento experimental sencillo y rápido para la generación de fibroblastos primarios de las orejas y la cola de los ratones. El procedimiento no requiere una formación especial de los animales y se puede utilizar para la generación de cultivos de fibroblastos de los oídos almacenados a temperatura ambiente durante hasta 10 días. 

Video: Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Stable and Efficient Genetic Modification of Cells in the Adult Mouse V-SVZ for the Analysis of Neural Stem Cell Autonomous and Non-autonomous Effects

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are restricted to two specific neurogenic niches: the subgranular zone of the dentate gyrus in the hippocampus and the ventricular-subventricular zone (V-SVZ; also called subependymal zone or SEZ) in the walls of the lateral ventricles. The development of in vivo gene transfer strategies for adult stem cell populations (i.e. those of the mammalian brain) resulting in long-term expression of desired transgenes in the stem cells and their derived progeny is a crucial tool in current biomedical and biotechnological research. Here, a direct in vivo method is presented for the stable genetic modification of adult mouse V-SVZ cells that takes advantage of the cell cycle-independent infection by LVs and the highly specialized cytoarchitecture of the V-SVZ niche. Specifically, the current protocol involves the injection of empty LVs (control) or LVs encoding specific transgene expression cassettes into either the V-SVZ itself, for the in vivo targeting of all types of cells in the niche, or into the lateral ventricle lumen, for the targeting of ependymal cells only. Expression cassettes are then integrated into the genome of the transduced cells and fluorescent proteins, also encoded by the LVs, allow the detection of the transduced cells for the analysis of cell autonomous and non-autonomous, niche-dependent effects in the labeled cells and their progeny.

Here we describe a procedure based on the use of lentiviral particles for the long-term genetic modification of neural stem cells and/or their adjacent ependymal cells in the adult ventricular-subventricular neurogenic niche which allows the separate analysis of cell autonomous and non-autonomous, niche-dependent effects on neural stem cells.

Estable y eficiente modificación genética de células en el ratón adulto V-SVZ para el análisis de neural de células madre Autónoma y los efectos no autónomos

Relatively quiescent somatic stem cells support life-long cell renewal in most adult tissues. Neural stem cells in the adult mammalian brain are ...

Investigación

Biología del Desarrollo

Murine Dermal Fibroblast Isolation by FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast physiology and pathology underlie a spectrum of clinical entities, including fibroses in multiple organs, hypertrophic scarring following burns, loss of cardiac function following ischemia, and the formation of cancer stroma. However, fibroblasts remain a poorly characterized type of cell, largely due to their inherent heterogeneity. Existing methods for the isolation of fibroblasts require time in cell culture that profoundly influences cell phenotype and behavior. Consequently, many studies investigating fibroblast biology rely upon in vitro manipulation and do not accurately capture fibroblast behavior in vivo. To overcome this problem, we developed a FACS-based protocol for the isolation of fibroblasts from the dorsal skin of adult mice that does not require cell culture, thereby preserving the physiologic transcriptional and proteomic profile of each cell. Our strategy allows for exclusion of non-mesenchymal lineages via a lineage negative gate (Lin-) rather than a positive selection strategy to avoid pre-selection or enrichment of a subpopulation of fibroblasts expressing specific surface markers and be as inclusive as possible across this heterogeneous cell type.

Fibroblast behavior underlies a spectrum of clinical entities, but they remain poorly characterized, largely due to their inherent heterogeneity. Traditional fibroblast research relies upon in vitro manipulation, masking in vivo fibroblast behavior. We describe a FACS-based protocol for the isolation of mouse skin fibroblasts that does not require cell culture.

Murino fibroblastos dérmicos aislamiento por FACS

Fibroblasts are the principle cell type responsible for secreting extracellular matrix and are a critical component of many organs and tissues. Fibroblast ...

Isolation of CD 90+ Fibroblast/Myofibroblasts from Human Frozen Gastrointestinal Specimens

Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and promotion of the tumor microenvironment. While there are currently many techniques for the isolation of MFs from gastrointestinal (GI) tissues, this protocol introduces a novel element of isolation of these stromal cells from frozen tissue. Freezing GI tissue specimens not only allows the researcher to acquire samples from worldwide collaborators, biobanks, and commercial vendors, it also permits the delayed processing of fresh samples. The described protocol will consistently yield characteristic spindle-shaped cells with the MF phenotype that express the markers CD90, &#945;-SMA and vimentin. As these cells are derived from patient samples, the use of primary cells also confers the benefit of closely mimicking MFs from disease states-namely cancer and inflammatory bowel diseases. This technique has been validated in gastric, small bowel, and colonic MF primary culture generation. Primary MF cultures can be used in a vast array of experiments over a number of passage and their purity assessed by both immunocytochemistry and flow cytometry analysis.

Here, a protocol to isolate and establish primary fibroblast/myofibroblast (MF) cultures from frozen gastric, small intestinal, and colonic tissue-yielding cells with a MF phenotype-is presented. These cells express CD90, &#945;-SMA and vimentin. MFs can be used for a variety of functional assays including enzymatic activity and cytokine production.

Aislamiento de CD 90+ fibroblastos / miofibroblastos de congelados Humanos gastrointestinales Especímenes


Fibroblasts/myofibroblasts (MFs) have been gaining increasing attention for their role in pathogenesis and their contributions to both wound healing and ...

Isolation and Differentiation of Adipose-Derived Stem Cells from Porcine Subcutaneous Adipose Tissues

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. Although numerous immortalized adipogenic cell lines have been established, adipose-derived stem cells from the stromal vascular fraction of subcutaneous white adipose tissues provide a reliable cellular system ex vivo much closer to adipose development in vivo. Pig adipose-derived stem cells (pADSC) are isolated from 7- to 9-day old piglets. The dorsal white fat depot of porcine subcutaneous adipose tissues is sliced, minced and collagenase digested. These pADSC exhibit strong potential to differentiate into adipocytes. Moreover, the pADSC also possess multipotency, assessed by selective stem cell markers, to differentiate into various mesenchymal cell types including adipocytes, osteocytes, and chondrocytes. These pADSC can be used for clarification of molecular switches in regulating classical adipocyte differentiation or in direction to other mesenchymal cell types of mesodermal origin. Furthermore, extended lineages into cells of ectodermal and endodermal origin have recently been achieved. Therefore, pADSC derived in this protocol provide an abundant and assessable source of adult mesenchymal stem cells with full multipotency for studying adipose development and application to tissue engineering of regenerative medicine.

This protocol describes the isolation of pig adipose-derived stem cells (pADSC) from subcutaneous adipose tissues with examination of multipotency. The multipotent pADSC are used to delineate processes of adipocyte differentiation and study transdifferentiation into multiple cell lineages of mesodermal mesenchyme or further lineages of ectoderm and endoderm for regenerative studies.

El aislamiento y la diferenciación de células madre derivadas de tejido adiposo subcutáneo de la especie porcina tejidos adiposos

Obesity is an unconstrained worldwide epidemic. Unraveling molecular controls in adipose tissue development holds promise to treat obesity or diabetes. ...

Using Primary Neurosphere Cultures to Study Primary Cilia

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, postnatal, and adult life. In addition, most differentiated neurons possess primary cilia that house signaling receptors, such as G-protein-coupled receptors, and signaling molecules, such as adenylyl cyclases. The primary cilium determines the activity of multiple developmental pathways, including the sonic hedgehog pathway during embryonic neuronal development, and also functions in promoting compartmentalized subcellular signaling during adult neuronal function. Unsurprisingly, defects in primary cilium biogenesis and function have been linked to developmental anomalies of the brain, central obesity, and learning and memory deficits. Thus, it is imperative to study primary cilium biogenesis and ciliary trafficking in the context of neural stem/progenitor cells and differentiated neurons. However, culturing methods for primary neurons require considerable expertise and are not amenable to freeze-thaw cycles. In this protocol, we discuss culturing methods for mixed populations of neural stem/progenitor cells using primary neurospheres. The neurosphere-based culturing methods provide the combined benefits of studying primary neural stem/progenitor cells: amenability to multiple passages and freeze-thaw cycles, differentiation potential into neurons/glia, and transfectability. Importantly, we determined that neurosphere-derived neural stem/progenitor cells and differentiated neurons are ciliated in culture and localize signaling molecules relevant to ciliary function in these compartments. Utilizing these cultures, we further describe methods to study ciliogenesis and ciliary trafficking in neural stem/progenitor cells and differentiated neurons. These neurosphere-based methods allow us to study cilia-regulated cellular pathways, including G-protein-coupled receptor and sonic hedgehog signaling, in the context of neural stem/progenitor cells and differentiated neurons.

The primary cilium is fundamentally important in neural progenitor cell proliferation, neuronal differentiation, and adult neuronal function. Here, we describe a method to study ciliogenesis and the trafficking of signaling proteins to cilia in neural stem/progenitor cells and differentiated neurons using primary neurosphere cultures.

El uso de cultivos de neuroesferas primarias estudian primaria cilios

The primary cilium is fundamentally important for the proliferation of neural stem/progenitor cells and for neuronal differentiation during embryonic, ...

Generation and Culturing of Primary Human Keratinocytes from Adult Skin

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. In addition, keratinocytes play an essential role in the initiation, maintenance, and regulation of epidermal immune responses by being part of the innate immune system responding to antigenic stimuli in a fast, nonspecific manner. Here, we describe a protocol for isolation of primary human keratinocytes from adult skin, and demonstrate that these cells respond to calcium-induced terminal differentiation, as measured by an increased expression of the differentiation marker involucrin. In addition, we show that the isolated keratinocytes are responsive to IL-1&#946;-induced activation of intracellular signaling pathways as measured by the activation of the p38 MAPK pathway. Taken together, we describe a method for isolation and culturing of primary human keratinocytes from adult skin. Because the keratinocytes are the predominant cell type in the epidermis, this method is useful to study molecular mechanisms in cutaneous biology in vitro.

The human skin acts as a first line of defense against the external environment. We present a method for isolating primary human keratinocytes from adult skin. These isolated keratinocytes are useful in numerous experimental setups, and are a highly suitable model for studying molecular mechanisms in cutaneous biology in vitro.

Generación y cultivo de Keratinocytes humanos primarios de piel adulto

The main function of keratinocytes is to provide the structural integrity of the epidermis, thereby maintaining a mechanical barrier to the outside world. ...

Direct Lineage Reprogramming of Adult Mouse Fibroblast to Erythroid Progenitors

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell fate determining and maturing factors. We previously set out to define the minimal set of factors necessary for instructing red blood cell development using direct lineage reprogramming of fibroblasts into induced erythroid progenitors/precursors (iEPs). We showed that overexpression of Gata1, Tal1, Lmo2, and c-Myc (GTLM) can rapidly convert murine and human fibroblasts directly to iEPs that resemble bona fide erythroid cells in terms of morphology, phenotype, and gene expression. We intend that iEPs will provide an invaluable tool to study erythropoiesis and cell fate regulation. Here we describe the stepwise process of converting murine tail tip fibroblasts into iEPs via transcription factor-driven direct lineage reprogramming (DLR). In this example, we perform the reprogramming in fibroblasts from erythroid lineage-tracing mice that express the yellow fluorescent protein (YFP) under the control of the erythropoietin receptor gene (EpoR) promoter, enabling visualization of erythroid cell fate induction upon reprogramming. Following this protocol, fibroblasts can be reprogrammed into iEPs within five to eight days. 
While improvements can still be made to the process, we show that GTLM-mediated reprogramming is a rapid and direct process, yielding cells with properties of bona fide erythroid progenitor and precursor cells.

Here we present our protocol for producing induced erythroid progenitors (iEPs) from mouse adult fibroblasts using transcription factor-driven direct lineage reprogramming (DLR).

Directo linaje reprogramación de fibroblastos de ratón adulto a progenitores eritroides

Erythroid cell commitment and differentiation proceed through activation of a lineage-restricted transcriptional network orchestrated by a group of cell ...

Tissue Preparation and Immunostaining of Mouse Craniofacial Tissues and Undecalcified Bone

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and simple protocol to detect protein expression during craniofacial morphogenesis/pathogenesis using mouse craniofacial tissues as examples. The protocol consists of preparation and cryosectioning of tissues, indirect immunofluorescence, image acquisition, and quantification. In addition, a method for preparation and cryosectioning of undecalcified hard tissues for immunostaining is described, using craniofacial tissues and long bones as examples. Those methods are key to determine the protein expression and morphological/anatomical changes in various tissues during craniofacial morphogenesis/pathogenesis. They are also applicable to other tissues with appropriate modifications. Knowledge of the histology and high quality of sections are critical to draw scientific conclusions from experimental outcomes. Potential limitations of this methodology include but are not limited to specificity of antibodies and difficulties of quantification, which are also discussed here.

Here, we present a detailed protocol to detect and quantify protein levels during craniofacial morphogenesis/pathogenesis by immunostaining using mouse craniofacial tissues as examples. In addition, we describe a method for preparation and cryosectioning of undecalcified hard tissues from young mice for immunostaining.

Preparación de tejidos e inmunomanchación de tejidos craneofaciales de ratón y hueso no descalcificado

Tissue immunostaining provides highly specific and reliable detection of proteins of interest within a given tissue. Here we describe a complete and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Disección, cultivo y análisis de células de cresta neural craneal primarias de ratón para el estudio de la delaminación y migración de células de cresta neuronal

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ...

Isolation and Differentiation of Primary Myoblasts from Mouse Skeletal Muscle Explants

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to differentiate into later stages of muscle development. The protocol provided here describes a robust method for the isolation and culture of a highly proliferative population of myoblast cells from young adult mouse skeletal muscle explants. These cells are useful for the study of the metabolic properties of skeletal muscle of different mouse models, as well as in other downstream applications such as transfection with exogenous DNA or transduction with viral expression vectors. The level of differentiation and metabolic profile of these cells depends on the length of exposure, and composition of the media used to induce myoblast differentiation. These methods provide a robust system for the study of mouse muscle cell metabolism ex vivo. Importantly, unlike in vivo models, the methods described here provide a cell population that can be expanded and studied with high levels of reproducibility.

Myoblasts are proliferating precursor cells that differentiate to form polynucleated myotubes and eventually skeletal muscle myofibers. Here, we present a protocol for efficient isolation and culture of primary myoblasts from young adult mouse skeletal muscles. The method enables molecular, genetic, and metabolic studies of muscle cells in culture.

Aislamiento y diferenciación de los mioblastos primarios de los explantes musculares esqueléticos de ratón

Primary myoblasts are undifferentiated proliferating precursors of skeletal muscle. They can be cultured and studied as muscle precursors or induced to ...