Name: generation of genetically modified organotypic skin cultures using devitalized human dermis
Uploaded: 2015-12-14T14:00:00.000+00:00
Duration: 9 min 16 s
Description: Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Este trabajo fue supportedby la Sociedad Americana del Cáncer de Investigación estudiosos Grant (RSG-12-148-01-DDC) y el Premio de Biología CIRM Básica (RB4-05779).

El protocolo de la piel humana se realizó de acuerdo con las directrices de la Universidad de California, Comité de Ética de Investigación de San Diego. La piel humana se puede obtener a partir de muestras quirúrgicas desechadas o comprado a los bancos de la piel (banco de piel aparece en la Tabla de materiales / Equipo). La ubicación donde la piel se deriva de o la edad del donante no es crítico para el experimento, siempre y cuando las proteínas de la membrana basal (colágeno / laminina) en la dermis no se degradan. 1. Preparación de desvitalizados Dermis Humanos <ol><li> Tras la recepción de la piel humana, colocar el tejido en la campana de cultivo de tejido para descongelar (~ 3-5 min). Dependiendo del tamaño de la piel, colocar el tejido descongelado en un tubo estéril desechable de 50 ml o 125 ml cónica botella. El tamaño típico de la piel desde el banco de piel es de 0,75 pies cuadrados. </li><li> Llene el recipiente de 75% de su capacidad con penicilina y estreptomicina 4x (penicilina / estreptomicina) en 1x PBS. Agitar vigorosamente durante5 min y deseche el líquido. Repita este paso 3 veces más. </li><li> Después del último lavado, transferir el tejido a un nuevo tubo / botella y añadir 4x fresco pen / strep / PBS para llenar 75% del contenedor. Incubar esta mezcla en una incubadora de cultivo de tejidos a 37 ° C durante 2 semanas para permitir la separación de la dermis de la epidermis. </li><li> En condiciones estériles, el uso de fórceps para despegar la epidermis de la dermis. La dermis es completamente blanco, mientras que la epidermis tendrán coloración en función de la carrera del donante (Figura 1A). Deseche la epidermis, según el protocolo de la institución para hacer frente a los tejidos humanos. </li><li> Coloque la dermis en un tubo o botella y lavarlo en 4x pen / strep diluido en PBS 1x con agitación vigorosa (5 min lavados por 4 veces). Después del último lavado, transferir la dermis a un nuevo tubo / botella en 4x pen / strep diluido en PBS 1x y se almacena a 4 ° C hasta su utilización. Dermis pueden almacenarse a 4 ° C durante un máximo de un año. </li></ol&gt; 2. Modificar genéticamente primaria queratinocitos humanos NOTA: Use las células phoenix anfotrópicos cultivadas en medio DMEM completo [DMEM + 10% de suero bovino fetal (FBS) + penicilina / estreptomicina] para producir virus para infectar los queratinocitos humanos primarios. Genes de empaquetamiento virales que codifican proteínas tales como gag-pol y el sobre se integran de manera estable en el genoma de la célula phoenix que permite la producción de virus cuando se transfecta con un vector retroviral. Células Phoenix se pueden transfectar con una alta eficiencia permitiendo para alta producción de título viral. Virus producido a partir de esta línea de envasado también puede infectar una gran variedad de células de mamífero incluyendo humanos. <ol><li> Día 1: El día antes de la transfección, las células Phoenix semillas en una placa de 6 pocillos a una densidad de 800.000 células por pocillo en medio DMEM completo. </li><li> Día 2: El día de la transfección, utilice 3 g de vectores retrovirales por pocillo. Alícuota 3 g de vector retroviral en un tubo de 1,5 ml. Use una mezcla de 100 μ; l DMEM más de 6 l de reactivo de transfección por cada 3 g de vector. Mezclar 6 l de reactivo de transfección con 100 l DMEM en un tubo de 1,5 ml. (Los vectores retrovirales que se utilizan se enumeran en la tabla de equipos / materiales). <ol><li> Incubar durante 5 min y añadir la mezcla de 106 l en el tubo de pre-alícuota que contiene el 3 g de vector retroviral. Mezclar e incubar a temperatura ambiente durante 30 min. Añadir gota a gota toda esta mezcla a cada pocillo de las células Phoenix. </li></ol></li><li> Día 3: El día después de la transfección, retire el medio de las células Phoenix y añadir 2 ml de medio DMEM fresca completa. El mismo día, la semilla queratinocitos humanos primarios en placas de 6 pocillos a una densidad de 75.000 células por pocillo. Mantener los queratinocitos en un medio de suero de queratinocitos libre (KCSFM) con antibióticos (penicilina / estreptomicina). NOTA: los queratinocitos humanos primarios fueron adquiridos. Las células se pueden comprar en una variedad de proveedores (que aparece en la tabla de materiales / Equipo). </li><li> Día 4: Harconferir los medios de comunicación de las células transfectadas phoenix, que ahora contiene partículas virales. Pase los medios de comunicación a través de un filtro de 0,45 micras con una jeringa (para eliminar cualquier célula phoenix contaminantes) y coloque 2 ml en los queratinocitos (chapados a 75.000 células / pocillo el día anterior: véase el paso 2.3). Añadir bromuro de hexadimetrina (5 g / ml) a los medios para ayudar a mediar en el proceso de infección. Los títulos virales son ~ 1 x 10 7 TU / ml. <ol><li> Haga girar las placas de 6 pocillos en una centrifugadora a 200 xg durante 1 hora a temperatura ambiente. Después de la escisión, retire el soporte que contiene el virus y lavar las células una vez con PBS 1x antes de añadir KCSFM. </li></ol></li><li> Día 5: infectar el mismo lote de queratinocitos de nuevo como en el paso 2.4. NOTA: Seleccionar los queratinocitos utilizando un fármaco si hay un marcador seleccionable en el vector retroviral y expandir para su uso en análisis de aguas abajo o aplicaciones tales como cultivos organotípicos. </li></ol> 3. Configuración de las Culturas de la piel organotípicos <ol><li> Construir elcasete organotípico a partir de materiales comunes que se encuentran en el laboratorio o las cintas puede ser por encargo a través de un taller de fabricación de metal (Figura 2 A - F). Para hacer estos cassettes de una placa de cultivo tisular 3,5 cm, utilizar un cauterio para eliminar un cuadrado de 1,0 cm x 1,0 cm desde el centro de la tapa de una placa de 3,5 cm (Figura 2A-B). Haga esto en condiciones estériles. <ol><li> Adjuntar clavijas cuadradas a la parte inferior de la casete (tapa de 3,5 cm plato) usando esmalte de uñas transparente (Figura 2C). Permitir 5 min para el esmalte de uñas se seque y voltear la casete de manera que está descansando sobre las clavijas cuadradas (Figura 2D). Coloque el casete en un plato de 6 cm. NOTA: Plaza clavijas se pueden comprar en una variedad de tiendas de artesanías. </li></ol></li><li> Preparar medio de crecimiento de queratinocitos (KGM) compuesta de 66% de DMEM, 22% F12 de Ham, 100 unidades / ml de penicilina, 100 mg / ml de estreptomicina, 10% de FBS, 2,67 g / ml adenina, 40 ng / ml de hidrocortisona, 10 la toxina del cólera ng / ml, 4,94 mg / ml de insulina, 5 mg / ml de transferrina, 1,36 ng / ml triyodo-L-tironina, 10 ng / ml EGF, y 10 mg / ml de hidrocloruro de ciprofloxacino. Filtra los medios de comunicación a través de un filtro y tienda de 0,22 micras a 4 ° C hasta su utilización. </li><li> Tome la dermis de la solución PBS 4x penicilina / estreptomicina + y lavar 2 veces en KGM y luego incubar en KGM a 37 ° C durante dos días antes de su uso. </li><li> Cortar la dermis utilizando un bisturí para 1,5 cm x 1,5 cm trozos de tamaño y luego se coloca en el cassette organotípico. Coloque la dermis con la parte superior de la dermis hacia arriba. La parte superior de la dermis será el lado que entra en contacto con los queratinocitos (Figura 1B). </li><li> Coloque la dermis pre-cortadas en el orificio cuadrado de la casete organotípicos con el lado superior de la dermis hacia arriba (Figura 3A). </li><li> Voltear todo el casete organotípico contiene la dermis sobre el uso de fórceps (Figura 3B). Descongele la solución de la matriz extracelular en el hielo. Use una aguja y una jeringa para extraer 100 l de solución de matriz extracelular por casete. Añadir 5 gotas a la parte inferior de la dermis (lado brillante) y agitar ligeramente para asegurar una distribución uniforme por toda la dermis (Figura 3B). </li><li> Permitir a 5 min de la solución comercial matriz extracelular para solidificar completamente (Figura 3C). Después de la solidificación, utilizar pinzas para voltear el casete hacia atrás. Añadir 4 ml de KGM al plato 6 cm. </li><li> Coloque 0,5-1 millones de queratinocitos modificados genéticamente en los cassettes que contienen organotípicos la dermis (Figura 3D). <ol><li> Trypsinize queratinocitos mediante la colocación de 4 ml de tripsina al 0,05% en placas de 10 cm durante 5 min y se inactiva con 4 ml de medio DMEM completo. </li><li> Contar los queratinocitos utilizando un hemocitómetro y luego girar hacia abajo a 200 xg durante 5 min. Resuspender las células 0,5-1.000.000 (dependiendo del número de células disponibles) en 90 μl de KGM y colocar todas las células en la dermis. NOTA: Es importante colocar el mismo número de células en la dermis dentro del mismo experimento para garantizar que las diferencias observadas en espesor de la epidermis regenerada no es debido a diferencias en el número de células de partida. Colocación de menos de 0,5 millones de células de la dermis puede conducir a una mala estratificación y diferenciación. </li></ol></li><li> Cambie medios KGM en los cultivos organotípicos cada dos días. Tejido Cosecha 1-14 días después de la colocación de los queratinocitos en la dermis. Estratificación completa y la diferenciación de la epidermis por lo general se produce después del día 5. </li></ol>

<ol>
	<li>Tadeu, A. M., Horsley, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+stem+cells+in+adult+skin.">Epithelial stem cells in adult skin.</a> Current topics in developmental biology. 107, 107-131 (2014).</li><li>Sen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remembering+one's+identity:+the+epigenetic+basis+of+stem+cell+fate+decisions.">Remembering one's identity: the epigenetic basis of stem cell fate decisions.</a> FASEB J. 25, 2123-2128 (2011).</li><li>Segre, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+barrier+formation+and+recovery+in+skin+disorders.">Epidermal barrier formation and recovery in skin disorders.</a> J Clin Invest. 116, 1150-1158 (2006).</li><li>Eckert, R. L., Sturniolo, M. T., Broome, A. M., Ruse, M., Rorke, E. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transglutaminase+function+in+epidermis.">Transglutaminase function in epidermis.</a> J Invest Dermatol. 124, 481-492 (2005).</li><li>Lopez-Pajares, V., Yan, K., Zarnegar, B. J., Jameson, K. L., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+pathways+in+disorders+of+epidermal+differentiation.">Genetic pathways in disorders of epidermal differentiation.</a> Trends Genet. 29, 31-40 (2013).</li><li>Fuchs, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidermal+differentiation:+the+bare+essentials.">Epidermal differentiation: the bare essentials.</a> J Cell Biol. 111, 2807-2814 (1990).</li><li>Green, H., Kehinde, O., Thomas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+of+cultured+human+epidermal+cells+into+multiple+epithelia+suitable+for+grafting.">Growth of cultured human epidermal cells into multiple epithelia suitable for grafting.</a> Proc Natl Acad Sci U S A. 76, 5665-5668 (1979).</li><li>Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+cancer+in+human+skin+tissue.">Modelling cancer in human skin tissue.</a> Nat Rev Cancer. 6, 270-280 (2006).</li><li>Parenteau, N. L., Bilbo, P., Nolte, C. J., Mason, V. S., Rosenberg, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+organotypic+culture+of+human+skin+keratinocytes+and+fibroblasts+to+achieve+form+and+function.">The organotypic culture of human skin keratinocytes and fibroblasts to achieve form and function.</a> Cytotechnology. 9, 163-171 (1992).</li><li>Oh, J. W., Hsi, T. C., Guerrero-Juarez, C. F., Ramos, R., Plikus, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organotypic+skin+culture.">Organotypic skin culture.</a> J Invest Dermatol. 133, e14 (2013).</li><li>Sen, G. L., 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=ZNF750+Is+a+p63+Target+Gene+that+Induces+KLF4+to+Drive+Terminal+Epidermal+Differentiation.">ZNF750 Is a p63 Target Gene that Induces KLF4 to Drive Terminal Epidermal Differentiation.</a> Dev Cell. 22, 669-677 (2012).</li><li>Sen, G. L., Webster, D. E., Barragan, D. I., Chang, H. Y., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+differentiation+in+a+self-renewing+mammalian+tissue+by+the+histone+demethylase+JMJD3.">Control of differentiation in a self-renewing mammalian tissue by the histone demethylase JMJD3.</a> Genes Dev. 22, 1865-1870 (2008).</li><li>Mistry, D. S., Chen, Y., Wang, Y., Zhang, K., Sen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SNAI2+controls+the+undifferentiated+state+of+human+epidermal+progenitor+cells.">SNAI2 controls the undifferentiated state of human epidermal progenitor cells.</a> Stem Cells. 32, 3209-3218 (2014).</li><li>Truong, A. B., Kretz, M., Ridky, T. W., Kimmel, R., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=p63+regulates+proliferation+and+differentiation+of+developmentally+mature+keratinocytes.">p63 regulates proliferation and differentiation of developmentally mature keratinocytes.</a> Genes Dev. 20, 3185-3197 (2006).</li><li>Kretz, 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=Control+of+somatic+tissue+differentiation+by+the+long+non-coding+RNA+TINCR.">Control of somatic tissue differentiation by the long non-coding RNA TINCR.</a> Nature. 493, 231-235 (2013).</li><li>Ridky, T. W., Chow, J. M., Wong, D. J., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Invasive+three-dimensional+organotypic+neoplasia+from+multiple+normal+human+epithelia.">Invasive three-dimensional organotypic neoplasia from multiple normal human epithelia.</a> Nat Med. 16, 1450-1455 (2010).</li><li>Mistry, D. S., Chen, Y., Sen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Progenitor+function+in+self-renewing+human+epidermis+is+maintained+by+the+exosome.">Progenitor function in self-renewing human epidermis is maintained by the exosome.</a> Cell Stem Cell. 11, 127-135 (2012).</li><li>Kretz, 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=Suppression+of+progenitor+differentiation+requires+the+long+noncoding+RNA+ANCR.">Suppression of progenitor differentiation requires the long noncoding RNA ANCR.</a> Genes Dev. 26, 338-343 (2012).</li><li>Mulder, K. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diverse+epigenetic+strategies+interact+to+control+epidermal+differentiation.">Diverse epigenetic strategies interact to control epidermal differentiation.</a> Nat Cell Biol. 14, 753-763 (2012).</li><li>Boxer, L. D., Barajas, B., Tao, S., Zhang, J., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ZNF750+interacts+with+KLF4+and+RCOR1,+KDM1A,+and+CTBP1/2+chromatin+regulators+to+repress+epidermal+progenitor+genes+and+induce+differentiation+genes.">ZNF750 interacts with KLF4 and RCOR1, KDM1A, and CTBP1/2 chromatin regulators to repress epidermal progenitor genes and induce differentiation genes.</a> Genes Dev. 28, 2013-2026 (2014).</li><li>Jameson, K. L., 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=IQGAP1+scaffold-kinase+interaction+blockade+selectively+targets+RAS-MAP+kinase-driven+tumors.">IQGAP1 scaffold-kinase interaction blockade selectively targets RAS-MAP kinase-driven tumors.</a> Nat Med. 19, 626-630 (2013).</li><li>Mistry, D. S., Chen, Y., Wang, Y., Sen, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+profiling+of+SNAI2+regulated+genes+in+primary+human+keratinocytes.">Transcriptional profiling of SNAI2 regulated genes in primary human keratinocytes.</a> Genomics data. 4, 43-46 (2015).</li><li>Mali, P., 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=RNA-guided+human+genome+engineering+via+Cas9.">RNA-guided human genome engineering via Cas9.</a> Science. 339, 823-826 (2013).</li><li>Doebis, 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=Efficient+in+vitro+transduction+of+epithelial+cells+and+keratinocytes+with+improved+adenoviral+gene+transfer+for+the+application+in+skin+tissue+engineering.">Efficient in vitro transduction of epithelial cells and keratinocytes with improved adenoviral gene transfer for the application in skin tissue engineering.</a> Transpl immunol. 9, 323-329 (2002).</li><li>Melo, S. P., 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=Somatic+correction+of+junctional+epidermolysis+bullosa+by+a+highly+recombinogenic+AAV+variant.">Somatic correction of junctional epidermolysis bullosa by a highly recombinogenic AAV variant.</a> Mol Ther. 22, 725-733 (2014).</li><li>Nanba, D., Matsushita, N., Toki, F., Higashiyama, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+expansion+of+human+keratinocyte+stem/progenitor+cells+carrying+a+transgene+with+lentiviral+vector.">Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector.</a> Stem cell res ther. 4, 127 (2013).</li><li>Sen, G. L., Reuter, J. A., Webster, D. E., Zhu, L., Khavari, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNMT1+maintains+progenitor+function+in+self-renewing+somatic+tissue.">DNMT1 maintains progenitor function in self-renewing somatic tissue.</a> Nature. 463, 563-567 (2010).</li><li>Shamir, E. R., Ewald, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+organotypic+culture:+experimental+models+of+mammalian+biology+and+disease.">Three-dimensional organotypic culture: experimental models of mammalian biology and disease.</a> Nat Rev Mol Cell Biol. 15, 647-664 (2014).</li><li>Krejci, N. C., Cuono, C. B., Langdon, R. C., McGuire, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+reconstitution+of+skin:+fibroblasts+facilitate+keratinocyte+growth+and+differentiation+on+acellular+reticular+dermis.">In vitro reconstitution of skin: fibroblasts facilitate keratinocyte growth and differentiation on acellular reticular dermis.</a> J Invest Dermatol. 97, 843-848 (1991).</li><li>Mathes, S. H., Ruffner, H., Graf-Hausner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+skin+models+in+drug+development.">The use of skin models in drug development.</a> Adv Drug Deliv Rev. 69-70, 81-102 (2014).</li></ol>

We have nothing to disclose.

La manipulación genética en la piel humana cultivos organotípicos ofrecen muchas ventajas para estudió comúnmente 2D células cultivadas, así como modelos de ratón. Culturas 2D carecen de las tres interacciones célula-matriz celular y extracelular de las células dimensionales que se encuentran en tejidos y órganos intactos. Estudios recientes también han encontrado enormes diferencias entre las células de cáncer de piel cultivadas en 2D y 3D con las células cultivadas en 3D que muestran mucho más similitu...

La epidermis humana es un epitelio estratificado que se conecta a la dermis subyacente a través de una matriz extracelular conocida como la epidermis terraza, plancha membrana basal no sólo sirve como una barrera impermeable para evitar la pérdida de humedad, sino también como una primera línea de defensa para proteger la cuerpo de sustancias extrañas y tóxicos 1. La capa basal, que es la capa más profunda de la epidermis, contiene las células madre epidérmicas y progenitoras que dan lugar a la progenie diferenciada que forman el resto de la epidermis 2. Como las células progenitoras se diferencian epidérmicas que migran hacia arriba para formar la primera capa de células diferenciadas conocida como la capa espinosa 3. En la capa espinosa, las células se convierten en la expresión de las queratinas 1 y 10, que a continuación proporciona la fuerza para soportar el estrés físico para las capas diferenciadas de la epidermis. Como las células de la capa espinosas diferenciar aún más, se mueven hacia arriba en la epidermis a FORm la capa granular, que se caracteriza por la formación de gránulos de queratohialina y laminares, así como proteínas estructurales que se ensamblan por debajo de la membrana plasmática. Como las células proceden en el proceso de diferenciación, las proteínas por debajo de la membrana plasmática son cruz vinculadas entre sí, mientras que los gránulos lamelares son extruidos a partir de las células para formar un lípido barrera rica llama el estrato córneo 4. Enfermedades que implican alteraciones en crecimiento epidérmico y el impacto diferenciación ~ 20% de la población 5. Por lo tanto, la comprensión de los mecanismos de este proceso es de gran importancia. Desde manifestación de muchas de estas enfermedades está supeditada a la célula-célula o célula-matriz de contacto, cultivos organotípicos donde la epidermis humana se reconstituye en un entorno 3D se han creado 6-10. Estos métodos implican típicamente el uso de queratinocitos primarios o transformadas sembradas sobre la matriz extracelular tales como humano desvitalizadodermis, Matrigel, o colágeno. Para entender los mecanismos de regulación de genes que son importantes en crecimiento epidérmico y la diferenciación, los queratinocitos se pueden manipular genéticamente a través de vectores retrovirales para derribar o sobreexpresar genes en cultivo 2D y luego reconstituirse en 3D. Estos métodos se han utilizado ampliamente para caracterizar genes implicados en la madre epidérmicas y células progenitoras auto-renovación y diferenciación, así como la progresión a neoplasia 11-21. Aquí, se proporciona un protocolo en profundidad sobre cómo alterar la expresión de genes en cultivos organotípicos epidérmicas a través del uso de los retrovirus.

<table><tbody><tr><td>Human skin</td><td>New York Firefighters Skin Bank</td><td>&lt;a href=&quot;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&quot;&gt;http://www.cornellsurgery.org/pro/services/burn-surgery/skin-bank.html&lt;/a&gt;</td><td></td></tr><tr><td>PEN/STREP</td><td>GIBCO</td><td>15140-122</td><td></td></tr><tr><td>amphotropic phoenix cell lines</td><td>ATCC</td><td>CRL-3213</td><td></td></tr><tr><td>FUGENE 6 transfection reagent</td><td>Promega</td><td>E2691</td><td></td></tr><tr><td>Keratinocyte Media (KCSFM)</td><td>Life Technologies</td><td>17005042</td><td></td></tr><tr><td>DMEM</td><td>GIBCO</td><td>11995</td><td></td></tr><tr><td>Ham&#039;s F12</td><td>Cambrex</td><td>12-615F</td><td></td></tr><tr><td>FBS</td><td>GIBCO</td><td>10437-028</td><td></td></tr><tr><td>Adenine</td><td>Sigma</td><td>A-9795</td><td></td></tr><tr><td>Cholera Toxin</td><td>Sigma</td><td>&amp;nbsp;C-8052</td><td></td></tr><tr><td>Hydrocortisone</td><td>Calbiochem</td><td>3896</td><td></td></tr><tr><td>Insulin</td><td>Sigma</td><td>I-1882</td><td></td></tr><tr><td>EGF</td><td>Invitrogen</td><td>13247-051</td><td></td></tr><tr><td>Transferrin&amp;nbsp;</td><td>Sigma</td><td>T-0665</td><td></td></tr><tr><td>Ciprofloxacin Hydrochloride</td><td>Serologicals</td><td>89-001-1</td><td></td></tr><tr><td>cautery</td><td>Bovie Medical Corporation</td><td>AA01</td><td></td></tr><tr><td>Matrigel</td><td>Corning</td><td>354234</td><td></td></tr><tr><td>Keratin 1 antibody</td><td>Biolegend</td><td>PRB-149P</td><td></td></tr><tr><td>square pegs</td><td>Arts and crafts stores</td><td></td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>ATCC</td><td>PCS-200-010</td><td></td></tr><tr><td>human neonatal keratinocytes</td><td>Cell Applications</td><td>102K-05n</td><td></td></tr><tr><td>MSCV retroviral vector</td><td>Clontech</td><td>634401</td><td></td></tr><tr><td>LZRS retroviral vector</td><td>Addgene</td><td></td><td></td></tr><tr><td>pSuper.Retro.Puro Retroviral vector</td><td>Oligoengine</td><td>VEC-PRT-0002&amp;nbsp;</td><td></td></tr><tr><td>hexadimethrine bromide&amp;nbsp;</td><td>Sigma</td><td>H9268-5G</td><td></td></tr></tbody></table>

generation of genetically modified organotypic skin cultures using devitalized human dermis

Los cultivos organotípicos permiten la reconstitución de un entorno 3D crítico para contacto célula-célula y célula-matriz interacciones que imita la función y la fisiología de sus homólogos de tejidos in vivo. Esto se ejemplifica con cultivos de piel organotípicos que recapitula fielmente la diferenciación epidérmica y el programa de la estratificación. Queratinocitos epidérmicos humanos primarios son genéticamente manipulable mediante retrovirus donde los genes pueden ser fácilmente sobreexpresan o derribados. Estos queratinocitos modificados genéticamente se pueden utilizar para regenerar la epidermis humana en cultivos de piel organotípicos proporcionan un poderoso modelo para estudiar vías genéticas de crecimiento epidérmico de impacto, la diferenciación y la progresión de la enfermedad. Los protocolos presentados aquí describen métodos para preparar dermis humana desvitalizados, así como para manipular genéticamente los queratinocitos humanos primarios a fin de generar cultivos de piel organotípicos. La piel humana regenerada se puede utilizar en downstream aplicaciones tales como perfiles de expresión génica, la inmunotinción y inmunoprecipitaciones cromatina seguido de secuenciación de alto rendimiento. Por lo tanto, la generación de estos cultivos de piel organotípicos modificados genéticamente permitirá la determinación de los genes que son críticos para el mantenimiento de la homeostasis de la piel.

El primer paso en la generación de la piel humana organotípicos es eliminar la epidermis de la dermis. La incubación de dos semanas de la piel en 37 ° C en 4x pen / strep / PBS debería permitir la separación de la dermis de la epidermis (Figura 1A). Si es difícil separar la epidermis y la dermis luego colocar el tejido a 37 ° C en 4x penicilina / estreptomicina / PBS durante una semana y luego tratar de pelar de nuevo utilizando fórceps. Una de las claves para la re...

Watch this Scientific Journal Video about Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis at JoVE.com

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Generación de Genéticamente Modificados cultivos de piel organotípicos Uso Desvitalizada Dermis Humana

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

generation-genetically-modified-organotypic-skin-cultures-using

Research

JoVE Journal

Developmental Biology

11.2K vistas.  UCSD Stem Cell Program, University of California, San Diego. The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis. 

Video: Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

A 3D Organotypic Melanoma Spheroid Skin Model

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased metastatic potential. Recently, mono-chemotherapies continue to improve by melanoma specific combination therapies with targeted kinase inhibitors. Still, metastatic melanoma remains a life-threatening disease because tumors exhibit primary resistance or develop resistance to novel therapies, thereby regaining tumorigenic capacity. In order to improve the therapeutic success of malignant melanoma, the determination of molecular mechanisms conferring resistance against conventional treatment approaches is necessary; however, it requires innovative cellular in vitro models. Here, we introduce an in vitro three-dimensional (3D) organotypic melanoma spheroid model that can portray the in vivo architecture of malignant melanoma and may warrant new insights into intra-tumoral as well as tumor-host interactions. The model incorporates defined numbers of mature and differentiated melanoma spheroids in a 3D human full skin reconstruction model consisting of primary skin cells. The cellular composition and differentiation status of the embedded melanoma spheroids is similar to the one of cutaneous melanoma metastasis in vivo. Using this organotypic melanoma spheroid model as a drug screening platform may support the identification of responders to selected combination therapies, while sparing the unnecessary treatment burden for non-responders, thereby increasing the benefit of therapeutic interventions.

Here, we present a protocol to generate a 3D organotypic melanoma spheroid skin model that recapitulates both the architecture and multicellular complexity of an organ/tumor in vivo but at the same time accommodates systematic experimental intervention.

Un modelo de piel Organotypic Melanoma 3D esferoide

Malignant transformation of melanocytes, the pigment cells of human skin, causes formation of melanoma, a highly aggressive cancer with increased ...

Investigación

Investigación sobre el cáncer

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. ...

Biología del Desarrollo

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

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Bioingeniería

Generation of 3D Skin Organoid from Cord Blood-derived Induced Pluripotent Stem Cells

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily functions. Biomimetics is the imitation of the models, systems, and elements of nature for the purpose of solving complex human problems1. Skin biomimetics is a useful tool for in vitro disease research and in vivo regenerative medicine. Human induced pluripotent stem cells (iPSCs) have the characteristic of unlimited proliferation and the ability of differentiation to three germ layers. Human iPSCs are generated from various primary cells, such as blood cells, keratinocytes, fibroblasts, and more. Among them, cord blood mononuclear cells (CBMCs) have emerged as an alternative cell source from the perspective of allogeneic regenerative medicine. CBMCs are useful in regenerative medicine because human leukocyte antigen (HLA) typing is essential to the cell banking system. We provide a method for the differentiation of CBMC-iPSCs into keratinocytes and fibroblasts and for generation of a 3D skin organoid. CBMC-iPSC-derived keratinocytes and fibroblasts have characteristics similar to a primary cell line. The 3D skin organoids are generated by overlaying an epidermal layer onto a dermal layer. By transplanting this 3D skin organoid, a humanized mice model is generated. This study shows that a 3D human iPSC-derived skin organoid may be a novel, alternative tool for dermatologic research in vitro and in vivo.

We propose a protocol that shows how to differentiate induced pluripotent stem cell-derived keratinocytes and fibroblasts and generate a 3D skin organoid, using these keratinocytes and fibroblasts. This protocol contains an additional step of generating a humanized mice model. The technique presented here will improve dermatologic research.

Generación de 3D piel organoide de cable derivadas de sangre inducida por las células de vástago Pluripotent

The skin is the body&#8217;s largest organ and has many functions. The skin acts as a physical barrier and protector of the body and regulates bodily ...

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern tissue engineering. A technical bottleneck is the deposition of collagen in vitro, as it is notoriously slow, resulting in sub-optimal formation of connective tissue and subsequent tissue cohesion, particularly in skin models. Here, we describe a method which involves the addition of differentially-sized sucrose co-polymers to skin cultures to generate macromolecular crowding (MMC), which results in a dramatic enhancement of collagen deposition. Particularly, dermal fibroblasts deposited a significant amount of collagen I/IV/VII and fibronectin under MMC in comparison to controls.
The protocol also describes a method to decellularize crowded cell layers, exposing significant amounts of extracellular matrix (ECM) which were retained on the culture surface as evidenced by immunocytochemistry. Total matrix mass and distribution pattern was studied using interference reflection microscopy. Interestingly, fibroblasts, keratinocytes and co-cultures produced cell-derived matrices (CDM) of varying composition and morphology. CDM could be used as &#34;bio-scaffolds&#34; for secondary cell seeding, where the current use of coatings or scaffolds, typically from xenogenic animal sources, can be avoided, thus moving towards more clinically relevant applications.
In addition, this protocol describes the application of MMC during the submerged phase of a 3D-organotypic skin co-culture model which was sufficient to enhance ECM deposition in the dermo-epidermal junction (DEJ), in particular, collagen VII, the major component of anchoring fibrils. Electron microscopy confirmed the presence of anchoring fibrils in cultures developed with MMC, as compared to controls. This is significant as anchoring fibrils tether the dermis to the epidermis, hence, having a pre-formed mature DEJ may benefit skin graft recipients in terms of graft stability and overall wound healing. Furthermore, culture time was condensed from 5 weeks to 3 weeks to obtain a mature construct, when using MMC, reducing costs.

Improving 2D and 3D Skin In Vitro Models Using Macromolecular Crowding

We present a protocol to obtain cell-derived matrices rich in extracellular matrix proteins, using macromolecular crowders (MMC). In addition, we present a protocol which incorporates MMC in 3D organotypic skin co-culture generation, which reduces culture time while maintaining maturity of construct.

La mejora en 2D y 3D de la piel<em&gt; In Vitro</em&gt; Uso de modelos macromoleculares hacinamiento

The glycoprotein family of collagens represents the main structural proteins in the human body, and are key components of biomaterials used in modern ...

Una revisión exhaustiva de los protocolos de cultivo de explantes de piel embrionaria aviar

Using Avian Skin Explants to Study Tissue Patterning and Organogenesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin explant cultures to examine different aspects of skin development are described. First, ex vivo organ cultures and manipulations offer researchers opportunities to observe and study the development of feather buds directly. Skin explant culture can grow for 7 days enabling direct analysis of cellular behavior and 4D imaging at intervals during this growth period. This also allows for physical and molecular manipulations of culture conditions to visualize tissue response. For example, growth factor-coated beads can be applied locally to induce changes in feather patterning in a limited area. Alternatively, viral transduction can be delivered globally in the culture media to up or downregulate gene expression. Second, the skin recombination protocol allows researchers to investigate tissue interactions between the epidermis and mesenchyme that are derived from different skin regions, different life stages, or different species. This affords an opportunity to test the time window in which the epithelium is competent to respond to signals and its ability to form different skin appendages in response to signals from different mesenchymal sources. Third, skin reconstitution using dissociated dermal cells overlaid with intact epithelium resets skin development and enables the study of the initial processes of periodic patterning. This approach also enhances our ability to manipulate gene expression among the dissociated cells before creating the reconstituted skin explant. This paper provides the three culture protocols and exemplary experiments to demonstrate their utility.

Autor destacado: Visión de las metodologías experimentales actuales de explantes de piel de ave

Here we describe protocols for three types of avian embryonic skin explant cultures that can be used to examine tissue interactions, 4D imaging timelapse movie (3D plus time), global or local perturbation of molecular function, and systems biology characterization.

Uso de explantes de piel de ave para estudiar el patrón de tejidos y la organogénesis

The developing avian skin during embryogenesis is a unique model that can provide valuable insights into tissue patterning. Here three variations on skin ...

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

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

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

El cultivo de una epidermis humana reconstruida tridimensional a gran escala

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

Generation of Self-assembled Vascularized Human Skin Equivalents

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to study skin development, wound healing, and grafting techniques. Many HSEs continue to lack vasculature and are additionally analyzed through post-culture histological sectioning which limits volumetric assessment of the structure. Presented here is a straightforward protocol utilizing accessible materials to generate vascularized human skin equivalents (VHSE); further described are volumetric imaging and quantification techniques of these constructs. Briefly, VHSEs are constructed in 12 well culture inserts in which dermal and epidermal cells are seeded into rat tail collagen type I gel. The dermal compartment is made up of fibroblast and endothelial cells dispersed throughout collagen gel. The epidermal compartment is made up of keratinocytes (skin epithelial cells) that differentiate at the air-liquid interface. Importantly, these methods are customizable based on needs of the researcher, with results demonstrating VHSE generation with two different fibroblast cell types: human dermal fibroblasts (hDF) and human lung fibroblasts (IMR90s). VHSEs were developed, imaged through confocal microscopy, and volumetrically analyzed using computational software at 4- and 8-week timepoints. An optimized process to fix, stain, image, and clear VHSEs for volumetric examination is described. This comprehensive model, imaging, and analysis techniques are readily customizable to the specific research needs of individual labs with or without prior HSE experience.

The goal of this protocol is to describe the generation and volumetric analysis of vascularized human skin equivalents using accessible and simple techniques for long term culture. To the extent possible, the rationale for steps is described to allow researchers the ability to customize based on their research needs.

Generación de equivalentes de piel humana vascularizada autoensamblada

Human skin equivalents (HSEs) are tissue engineered constructs that model epidermal and dermal components of human skin. These models have been used to ...

Generación de Genéticamente Modificados cultivos de piel organotípicos Uso Desvitalizada Dermis Humana

Resumen

Explorar más videos

Capítulos en este video

Un modelo de piel Organotypic Melanoma 3D esferoide

Generación y cultivo de Keratinocytes humanos primarios de piel adulto

Generación de una tridimensional grosor de la piel completa y automatizada Equivalente Heridas

Generación de 3D piel organoide de cable derivadas de sangre inducida por las células de vástago Pluripotent

La mejora en 2D y 3D de la piel

Uso de explantes de piel de ave para estudiar el patrón de tejidos y la organogénesis

Uso de explantes de piel de ave para estudiar el patrón de tejidos y la organogénesis

Uso de explantes de piel de ave para estudiar el patrón de tejidos y la organogénesis

El cultivo de una epidermis humana reconstruida tridimensional a gran escala

Generación de equivalentes de piel humana vascularizada autoensamblada