Los autores desean agradecer a Sally Roberts (Universidad de Birmingham, Birmingham, Reino Unido) para la especie de regalo del anticuerpo E1E4. Este trabajo fue apoyado por una subvención del Instituto Nacional del Cáncer (4R00CA137160-03).

1. Preparación para las Culturas Raft organotípicos <ol><li> Las rejillas balsa de metal mucho primero se trata con ácido crómico sulfúrico para eliminar cualquier residuo que pudiera interferir con el proceso de diferenciación. Sumergir rejillas metálicas en un vaso de precipitados de vidrio que contenía ácido sulfúrico durante una hora, a continuación, de forma continua durante la noche enjuaga con agua del grifo. Después de la noche a la mañana enjuague, las redes de balsas debe enjuagarse durante 3-5 horas en agua doblemente destilada. </li><li> Para prestar apoyo a las balsas, doble tres lados de la balsa de 0,5 cm en la misma distancia el uno del otro. Las rejillas de metal, entonces deben ser esterilizados en autoclave. </li><li> Preparar el tampón de reconstitución 10 veces mediante la adición de 2,2 g de NaHCO3 y 4,8 g de Hepes. Disolver en 100 ml de 0,05 M de NaOH. Filtrar esterilizar y almacenar en alícuotas de 5 ml a -20 ° C. </li><li> Prepare el 10 veces DMEM sin bicarbonato de sodio. Filtrar esterilizar y almacenar en alícuotas de 5 ml a -20 ° C. </li><li> Preparar el factor de crecimiento epidérmico del ratón: Disolver 100 mg de EGF y 10 mg de BSA cada en 10 ml de agua desionizada ddH 2 0. Combinar EGF y BSA y añadir a 80 ml ​​de agua desionizada ddH 2 0 para un volumen total de 100 ml. Filtrar a esterilizar, alícuota (5 ml) y en la tienda de -20 ° C. Para cada litro de medio E añadir 5 ml de 1 mg / ml de EGF (concentración final de 5 ng / ml). </li></ol> 2. Preparación de geles de colágeno <ol><li> Un gel de colágeno se requiere para cada balsa. El colágeno se debe mantener en hielo para evitar que se solidifique. Cada gel de colágeno requiere 3 ml de mezcla de colágeno que consta de: 2,4 ml de colágeno de tipo frío de cola de rata I (concentración final de 3-4 mg / ml), 0,3 ml de tampón 10X de la reconstitución, 0,3 ml de DMEM 10X and1-2 X 10 6 ratón J2 3T3 fibroblastos como células alimentadoras. Para soluciones de alta concentración de colágeno de cola de rata, se diluye en 0,02 M de ácido acético para conseguir la concentración de trabajo correcta. </li><li> Determinar el número de geles de colágeno (balsas) que se necesita y luego calcular una mezcla maestra de la cantidad de colágeno, reconstit 10 veceslución tampón, 10X DMEM y fibroblastos J2 requerido. Incluir suficiente en cada mezcla maestra para 3-4 geles adicionales debido a la viscosidad del colágeno. </li><li> Para preparar el gel de colágeno, trypsinize los fibroblastos J2 y neutralizar con el medio. Combine todos los fibroblastos y el lugar en un frasco cónico de 50 ml. Cuenta las células para determinar el número de balsas que se pueden hacer. De nuevo, 1-2x10 6 fibroblastos se requieren por gel de colágeno y, por tanto por balsa. Gira a las células a baja velocidad. </li><li> Aspirar el medio de la pastilla de fibroblastos y resuspender en la cantidad apropiada de tampón de reconstitución 10X, 10X DMEM y colágeno para el número de balsas calculados. Añadir el colágeno pasado para evitar que el gel se solidifique demasiado rápidamente. Una vez que el colágeno se añade, mezclar rápidamente, pero con cuidado para evitar la introducción de burbujas en el gel. El gel debe ser de un color naranja rojizo, indicativo de que el pH correcto. Si la mezcla de gel es demasiado amarillo, añadir un par de gotas de 1N, esterilizada por filtraciónNaOH y mezclar suavemente hasta obtener el color correcto. </li><li> Añadir 3 ml de la colágeno: mezcla de fibroblastos a los pocillos de una placa de cultivo de 6 pocillos. La mezcla de colágeno se pipeteó lentamente por el lado de cada pocillo para evitar la generación de burbujas. Colocar la placa en un 37 ° C tejido incubadora cultura y dejar que se solidifique durante 30 minutos. </li><li> Después de 30 minutos, añadir 3 ml de medio de E con EGF a la parte superior del gel de colágeno solidificado y devolver la placa a la incubadora. Los geles se deben mantener a 37 ° C durante al menos un día, pero utiliza un plazo de cuatro días. Los resultados óptimos se producen después de dos días de incubación. </li></ol> 3. Los queratinocitos Preparación para la diferenciación <ol><li> Queratinocitos de bajo pasaje debe ser aumentado a aproximadamente el 70% de confluencia. Cada balsa requiere 1-2x10 6 queratinocitos. Trypsinize los queratinocitos y neutralizar con un medio electrónico con EGF. Cuenta las células para determinar el número de balsas que se pueden hacer. Gira por las células en espe bajaed. Resuspender el precipitado en 1 ml de medio de E junto con la FGE para cada cultivo en balsas calculado. </li><li> Aspirar el medio de los geles de colágeno por la inclinación de la placa de 6 pocillos. Añadir 2 ml de medio fresco E con EGF a cada gel. Para cada gel, añadir cuidadosamente 1 ml de queratinocitos resuspendidos a los 2 ml de medio E ya presente en el pozo. Suavemente pipeta los queratinocitos más el 2 ml de los medios y luego dejar caer gota a gota de nuevo en el gel. Agitar o balanceo de la placa puede resultar en una distribución desigual de las células a un lado del gel de colágeno. Colocar la placa en la incubadora a 37 ° C. </li><li> Las células se cultivan hasta la confluencia, reemplazando el medio E diariamente. Las células son confluentes cuando los cambios en el medio a amarillo un día después de un cambio en el medio. Esto suele tardar entre 2-4 días. Si el medio no ha cambiado por día cuatro, proceder al paso siguiente. </li></ol> 4. Realización de las Culturas Raft <ol><li> Use pinzas estériles para colocar una rejilla metálica balsa de estériles, se inclinó por los lados, En un plato de 10 cm. Aspirar el medio de gel de colágeno. Para aflojar el gel de las paredes del pozo, ir alrededor del perímetro del gel con una espátula estéril usando un movimiento ascendente y descendente. Para quitar el gel de colágeno, incline ligeramente la placa y levantar mediante la colocación de una espátula por debajo. Colocar el gel de colágeno en la rejilla metálica sin generar burbujas entre la rejilla y el gel. Dos geles se pueden colocar en cada cuadrícula. </li><li> Con el fin de hacer una interfase aire-líquido, se añade medio de E sin EGF lentamente a la parte inferior del plato por lo que la rejilla balsa está en contacto con los medios pero el gel de colágeno no lo es. Los medios de comunicación no deben entrar a través de los agujeros en la red. Añadir poco a poco el medio para evitar la generación de burbujas en la red, ya que esto evitará que la diferenciación uniforme. Incubar las balsas a 37 ° C y cambiar el medio de cada dos días manteniendo la interfase aire-líquido. Coseche las balsas después de 14 días. </li></ol> 5. Los resultados representativos Cuando tl protocolo se realiza correctamente con queratinocitos primarios, este procedimiento se traducirá en un epitelio bien diferenciado donde las diferentes capas de la piel son evidentes. Esto se logrará mediante el examen histológico mediante la tinción de las secciones de las culturas balsa de hematoxilina y eosina (H &amp; E), como se muestra en la Figura 1B. Tinción H &amp; E también es útil para examinar la morfología de las balsas y el efecto de los virus o compuestos antivirales sobre la capacidad de las células para estratificar y diferenciar. Por ejemplo, balsas hechas de VPH-inmortalizado queratinocitos humanos primarios (HFK-31) son notablemente más gruesa en comparación con balsas hechas de queratinocitos normales de prepucio humano (HFK), lo que refleja un aumento en la tasa de proliferación y la capacidad de las proteínas del VPH para mantener las células de diferenciación activa en el ciclo celular (Figura 1B) 10. Esto da como resultado la retención de los núcleos a través del epitelio, mientras que los queratinocitos normales salen del ciclo celular en la diferenciación, resultando en una ruptura delenvoltura nuclear. Para examinar más de cerca la diferenciación, inmunohistoquímica se puede realizar para examinar marcadores específicos de diferenciación, incluyendo citoqueratinas, así como involucrina, y filagrina. Citoqueratinas diferentes se expresan en etapas específicas de la diferenciación 7. Como se muestra en la Figura 2, citoqueratina 10 (K10) no se expresa en la capa basal, y se encuentra sólo en las capas suprabasales que consisten en células progresivamente diferenciadores. En las balsas de VPH positivos, la expresión de K10 se retrasa en comparación con la de queratinocitos normales, reflejando la influencia de las proteínas del VPH en la diferenciación de los queratinocitos. Inmunohistoquímica también se puede utilizar para examinar el perfil de expresión de los genes virales. Se muestra en la Figura 3 es una tinción representativa examinar la expresión de la proteína E1 VPH ^ E4, que es una proteína finales cuya expresión está restringida a las capas superiores del epitelio. Como era de esperar, no hay manchas que se observa en las culturas balsa de lo normal Human queratinocitos prepucio. <img alt="Figura 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> Figura 1. Un esquema) de un método para la preparación de cultivos organotípicos balsa a partir de queratinocitos primarios o líneas de células epiteliales. B) Hematoxilina y eosina de las culturas balsa organotípicos generados a partir de queratinocitos de prepucio humano de forma estable el mantenimiento de VPH-31 (HFK-31) episomas, o de los queratinocitos normales de prepucio humano (HFK). Las capas epiteliales individuales se identifican, así como el tapón de colágeno. <img alt="Figura 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> Figura 2. Citoqueratina 10 (K10) expresión está restringida a las capas suprabasales del epitelio. Inmunohistoquímica se realizó en las secciones transversales de cultivos balsa organotípicos generados a partir de células HFK-31, así como HFKs normales usando un anticuerpo a K10. ADN celular se counterstained con DAPI. Las imágenes fueron capturadas utilizando confmicroscopía de fluorescencia vecinal. Las flechas indican la capa basal del epitelio. <img alt="Figura 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> Figura 3. E1 VPH ^ proteína E4 se produce al final de la fase productiva del ciclo de vida viral. Inmunohistoquímica se realizó en las secciones transversales de cultivos balsa organotípicos generados a partir de células HFK-31, así como HFKs normales usando anticuerpos para E1E4. ADN celular se counterstained con DAPI. Las imágenes fueron capturadas utilizando un microscopio de fluorescencia confocal. Las flechas indican la capa basal del epitelio.

<ol>
	<li>Andrei, G., Duraffour, S., VandenOord, J., Snoeck, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial+raft+cultures+for+investigations+of+virus+growth,+pathogenesis+and+efficacy+of+antiviral+agents.">Epithelial raft cultures for investigations of virus growth, pathogenesis and efficacy of antiviral agents.</a> Antiviral Research. 85, 431-449 (2010).</li><li>Asselineau, D., Prunieras, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+'simplified'+skin:+control+of+fabrication.">Reconstruction of 'simplified' skin: control of fabrication.</a> The British Journal of Dermatology. 111, 219-222 (1984).</li><li>Bodily, J., Alam, S., Chen, H., Meyers, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Organotypic epithelial raft cultures and the study of the natural history of human papillomavirus. , (2006).</li><li>Cheng, S., Schmidt-Grimminger, D. C., Murant, T., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation-dependent+up-regulation+of+the+human+papillomavirus+E7+gene+reactivates+cellular+DNA+replication+in+suprabasal+differentiated+keratinocytes.">Differentiation-dependent up-regulation of the human papillomavirus E7 gene reactivates cellular DNA replication in suprabasal differentiated keratinocytes.</a> Genes &#38; Development. 9, 2335-2349 (1995).</li><li>Dollard, S. C., Wilson, J. L., Demeter, L. M., Bonnez, W. R., Reichman, C., Broker, T. R., Chow, L. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+human+papillomavirus+and+modulation+of+the+infectious+program+in+epithelial+raft+cultures.+OFF.">Production of human papillomavirus and modulation of the infectious program in epithelial raft cultures. OFF.</a> Genes &#38; Development. 6, 1131-1142 (1992).</li><li>Frattini, M. G., Lim, H. B., Laimins, 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=In+vitro+synthesis+of+oncogenic+human+papillomaviruses+requires+episomal+genomes+for+differentiation-dependent+late+expression.">In vitro synthesis of oncogenic human papillomaviruses requires episomal genomes for differentiation-dependent late expression.</a> Proceedings of the National Academy of Sciences of the United States of America. 93, 3062-3067 (1996).</li><li>Kopan, R., Traska, G., 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=Retinoids+as+important+regulators+of+terminal+differentiation:+examining+keratin+expression+in+individual+epidermal+cells+at+various+stages+of+keratinization.">Retinoids as important regulators of terminal differentiation: examining keratin expression in individual epidermal cells at various stages of keratinization.</a> The Journal of Cell Biology. 105, 427-440 (1997).</li><li>Longworth, M. S., Laimins, 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=Pathogenesis+of+human+papillomaviruses+in+differentiating+epithelia.">Pathogenesis of human papillomaviruses in differentiating epithelia.</a> Microbiology and Molecular Biology Review. 68, 362-372 (2004).</li><li>Meyers, C., Frattini, M. G., Hudson, J. B., Laimins, 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=Biosynthesis+of+human+papillomavirus+from+a+continuous+cell+line+upon+epithelial+differentiation.">Biosynthesis of human papillomavirus from a continuous cell line upon epithelial differentiation.</a> Science. 257, 971-973 (1992).</li><li>Wilson, R., Laimins, 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=.">.</a> Differentiation of HPV-Containing Cells Using Organotypic Raft Culture or Methylcellulose. , 119-119 (2006).</li><li>zur Hausen, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Papillomavirus+infections--a+major+cause+of+human+cancers.">Papillomavirus infections--a major cause of human cancers.</a> Biochimica et Biophysica Acta. 1288, F55-F78 (1996).</li></ol>

No tenemos nada que revelar.

Se describe aquí un método que puede utilizarse para estudiar la diferenciación epitelial en general, pero también puede ser fácilmente adaptado para estudiar la patogénesis viral, así como la eficacia de la terapéutica potencial. Muchos virus orientar las células epiteliales, ya sea como el sitio primario de la infección, como en el caso de VPH, o en algún punto del ciclo de vida viral, como con herpesvirus. Aunque cada vez más cultivos organotípicos balsa es mucho tiempo, la capacidad de hacer una recapit...

<table border="1"><tbody><tr><td> Nombre de Reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Cola de Rata colágeno tipo 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> DMEM sin ​​NaHC0 3 </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> NaHC0 3 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> De acero inoxidable rejillas de metal </td><td> Williams y temple Co. </td><td> CR-03063-040-100-S </td></tr><tr><td> E medio 11 </td><td></td><td></td></tr><tr><td> Factor de Crecimiento Epidérmico </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

El desarrollo de las culturas organotípicos balsa epiteliales ha proporcionado a los investigadores con un eficiente sistema in vitro que fielmente recapitula la diferenciación epitelial. Hay muchos usos para este sistema. Por ejemplo, la capacidad de crecer cultivos tridimensionales balsa organotípicos de queratinocitos ha sido un hito importante en el estudio de los virus del papiloma humano (VPH) 1. El ciclo de vida del HPV está estrechamente vinculada a la diferenciación del epitelio escamoso 2. Organotípicos culturas balsa epiteliales como se ha demostrado aquí reproducir el ciclo de vida completo del virus del papiloma, incluidos los virus de la producción 3,4,5. Además, estos cultivos presentan lesiones displásicas balsas similares a los observados en la infección in vivo con el VPH. Por tanto, este sistema también puede utilizarse para estudiar los cánceres de células epiteliales, así como el efecto de fármacos sobre la diferenciación de células epiteliales en general. Originalmente desarrollado por Asselineau y Prunieras <shasta&gt; 6 y modificado por Kopan et al. 7, el sistema organotípico epitelial cultivo en balsas ha madurado en un modelo general, la cultura relativamente fácil, que implica el crecimiento de células sobre el colágeno se enchufa mantuvo a una interfase aire-líquido (Figura 1A). Durante el curso de 10-14 días, las células estratificar y diferenciar, formando un epitelio de grosor completo que produce específicos diferenciación-citoqueratinas. Balsas cosechadas se pueden examinar histológicamente, así como por las técnicas estándar de moleculares y bioquímicos. En este artículo, se describe un método para la generación de cultivos primarios balsa de queratinocitos humanos. La misma técnica se puede utilizar con las líneas celulares establecidas epiteliales, y puede ser fácilmente adaptado para su uso con el tejido epitelial de biopsias normales o enfermas 8. Muchos virus objetivo, ya sea el epitelio cutáneo o mucoso, como parte de su ciclo de vida replicativa. En los últimos años, la posibilidad de utilizar culto balsa organotípicoUres como un método de estudio de virus-huésped interacciones celulares se ha demostrado por varios herpesvirus, así como adenovirus, parvovirus, y poxvirus 9. Cultivos organotípicos balsa por lo tanto se puede adaptar para examinar la patogénesis viral, y son el único medio para poner a prueba nuevos agentes antivirales para los virus que no son cultivables en líneas celulares permanentes.

Watch this Scientific Journal Video about Generation of Organotypic Raft Cultures from Primary Human Keratinocytes at JoVE.com

Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

Un<em&gt; In vitro</em&gt; Método para imitar<em&gt; En vivo</emLa diferenciación&gt; epitelial se ha descrito. Muchos virus dirigirse a las células epiteliales como parte de su ciclo de vida del virus, y este método proporciona una manera de examinar el virus: las interacciones del huésped que se asemeja más a lo que ocurre<em&gt; En vivo</em&gt;. Esta técnica se puede utilizar con queratinocitos primarios, líneas celulares establecidas, así como biopsia de tejido normal o enfermo.

Generación de las Culturas Raft organotípicos de queratinocitos humanos primarios

The development of organotypic epithelial raft cultures has provided researchers with an efficient in vitro system that faithfully recapitulates ...

generation-organotypic-raft-cultures-from-primary-human

Research

JoVE Journal

Immunology and Infection

19.1K vistas.  University of North Carolina-Chapel Hill. Un In vitro Método para imitar En vivo epitelial se ha descrito. Muchos virus dirigirse a las células epiteliales como parte de su ciclo de vida del virus, y este método proporciona una manera de examinar el virus: las interacciones del huésped que se asemeja más a lo que ocurre En vivo. Esta técnica se puede utilizar con queratinocitos primarios, líneas celulares establecidas, así como biopsia de teji...

Video: Generation of Organotypic Raft Cultures from Primary Human Keratinocytes

A Simple and Efficient Method to Isolate Macrophages from Mixed Primary Cultures of Adult Liver Cells

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although the isolation methods of liver macrophages have been well-described4-6, most of these methods require sophisticated equipment, such as a centrifugal elutriator and technical skills. Here, we provide a novel method to obtain liver macrophages in sufficient number and purity from mixed primary cultures of adult rat liver cells, as schematically illustrated in Figure 1.
After dissociation of the liver cells by two-step perfusion method7,8,a fraction mostly composed of parenchymal hepatocytes is prepared and seeded into T75 tissue culture flasks with culture medium composed of DMEM and 10% FCS.Parenchymal hepatocytes lose the epithelial cell morphology within a few days in culture, degenerate or transform into fibroblast-like cells (Figure 2). As the culture proceeds, around day 6, phase contrast-bright, round macrophage-like cells start to proliferate on the fibroblastic cell sheet (Figure 2). The growth of the macrophage-like cells continue and reach to maximum levels around day 12, covering the cell sheet on the flask surface. By shaking of the culture flasks, macrophages are readily suspended into the culture medium. Subsequent transfer and short incubation in plastic dishes result in selective adhesion of macrophages(Figure 3), where as other contaminating cells remain suspended. After several rinses with PBS, attached macrophages are harvested. More than 106 cells can be harvested repeatedly from the same T75 tissue culture flask at two to three day intervals for more than two weeks(Figure 3).The purities of the isolated macrophages were 95 to 99%, as evaluated by flow cytometry or immunocytochemistry with rat macrophage-specific antibodies (Figure 4).The isolated cells show active phagocytosis of polystylene beads (Figure 5), proliferative response to recombinant GM-CSF, secretion of inflammatory/anti-inflammatory cytokines upon stimulation with LPS, and formation of multinucleated giant cells9.
In conclusion, we provide a simple and efficient method to obtain liver macrophages in sufficient number and purity without complex equipment and skills.This method might be applicable to other mammalian species.

A novel method to obtain macrophages from primary culture of rat liver cells is described. This method utilizes the proliferation of macrophages in the culture, followed by shaking of culture flasks and purification by selective attachment to plastic dishes. This technique efficiently provides liver macrophages without complex equipment and skills.

Un método simple y eficaz para aislar los macrófagos de la Mezcla de cultivos primarios de células de hígado en adultos

Kupffer cells are liver-specific resident macrophages and play an important role in the physiological and pathological functions of the liver1-3. Although ...

Investigación

Inmunología e Infección

Generation of Induced Regulatory T Cells from Primary Human Na&iuml;ve and Memory T Cells

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in immunologic homeostasis with the vast amount of self and commensal antigens in and on the human body. Perturbations in the balance between Tregs and inflammatory conventional T cells can result in immunopathology or cancer. Although therapeutic injection of Tregs has been shown to be efficacious in murine models of colitis1 , type I diabetes2 , rheumatoid arthritis and graft versus host disease,4 several fundamental differences in human versus mouse Treg biology5 has thus far precluded clinical use. The lack of sufficient number, purity, stability and homing specificity of therapeutic Tregs necessitated a dynamic platform of human Treg development on which to optimize conditions for their ex vivo expansion6.
Here we describe a method for the differentiation of induced Tregs (iTregs) from a single human peripheral blood donor which can be broken down into four stages: isolation of peripheral blood mononuclear cells, magnetic selection of CD4+ T cells, in vitro cell culture and fluorescence activated cell sorting (FACS) of T cell subsets. Since the Treg signature transcription factor forkhead box P3 (FoxP3) is an activation-induced transcription factor in humans7 and no other unique marker exists, a combinatorial panel of markers must be used to identify T cells with suppressor activity. After six days in culture, cells in our system can be demarcated into na&iuml;ve T cells, memory T cells or iTregs based on their relative expression of CD25 and CD45RA. As memory and na&iuml;ve T cells have different reported polarization requirements and plasticities8 , pre-sorting of the initial T cell population into CD45RA+ and CD45RO+ subsets can be used to examine these discrepancies. Consistent with others, our CD25HiCD45RA- iTregs express high levels of FoxP39 , GITR and CTLA-411 and low levels of CD12712 . Following FACS of each population, resultant cells can be used in a suppressor assay which evaluates the relative ability to retard the proliferation of carboxyfluorescein succinimidyl ester (CFSE)-labeled autologous T cells.

Generation of Induced Regulatory T Cells from Primary Human Na&#239;ve and Memory T Cells

We describe a method for generating regulatory, memory and na&#239;ve T cells from a single human blood donor. Polarized Tregs can be then compared to other subsets in a variety of genetic and functional applications with genetic homogeneity, including a suppression assay also detailed here.

La generación de células T reguladoras inducidas de células humanas primarias T vírgenes y de memoria

The development and maintenance of immunosuppressive CD4+ regulatory T cells (Tregs) contribute to the peripheral tolerance needed to remain in ...

Primary Microglia Isolation from Mixed Glial Cell Cultures of Neonatal Rat Brain Tissue

Microglia account for approximately 12% of the total cellular population in the mammalian brain. While neurons and astrocytes are considered the major cell types of the nervous system, microglia play a significant role in normal brain physiology by monitoring tissue for debris and pathogens and maintaining homeostasis in the parenchyma via phagocytic activity 1,2. Microglia are activated during a number of injury and disease conditions, including neurodegenerative disease, traumatic brain injury, and nervous system infection 3. Under these activating conditions, microglia increase their phagocytic activity, undergo morpohological and proliferative change, and actively secrete reactive oxygen and nitrogen species, pro-inflammatory chemokines and cytokines, often activating a paracrine or autocrine loop 4-6. As these microglial responses contribute to disease pathogenesis in neurological conditions, research focused on microglia is warranted.
	Due to the cellular heterogeneity of the brain, it is technically difficult to obtain sufficient microglial sample material with high purity during in vivo experiments. Current research on the neuroprotective and neurotoxic functions of microglia require a routine technical method to consistently generate pure and healthy microglia with sufficient yield for study. We present, in text and video, a protocol to isolate pure primary microglia from mixed glia cultures for a variety of downstream applications. Briefly, this technique utilizes dissociated brain tissue from neonatal rat pups to produce mixed glial cell cultures. After the mixed glial cultures reach confluency, primary microglia are mechanically isolated from the culture by a brief duration of shaking. The microglia are then plated at high purity for experimental study.
	The principle and protocol of this methodology have been described in the literature 7,8. Additionally, alternate methodologies to isolate primary microglia are well described 9-12. Homogenized brain tissue may be separated by density gradient centrifugation to yield primary microglia 12. However, the centrifugation is of moderate length (45 min) and may cause cellular damage and activation, as well as, cause enriched microglia and other cellular populations. Another protocol has been utilized to isolate primary microglia in a variety of organisms by prolonged (16 hr) shaking while in culture 9-11. After shaking, the media supernatant is centrifuged to isolate microglia. This longer two-step isolation method may also perturb microglial function and activation. We chiefly utilize the following microglia isolation protocol in our laboratory for a number of reasons: (1) primary microglia simulate in vivo biology more faithfully than immortalized rodent microglia cell lines, (2) nominal mechanical disruption minimizes potential cellular dysfunction or activation, and (3) sufficient yield can be obtained without passage of the mixed glial cell cultures.
	It is important to note that this protocol uses brain tissue from neonatal rat pups to isolate microglia and that using older rats to isolate microglia can significantly impact the yield, activation status, and functional properties of isolated microglia. There is evidence that aging is linked with microglia dysfunction, increased neuroinflammation and neurodegenerative pathologies, so previous studies have used ex vivo adult microglia to better understand the role of microglia in neurodegenerative diseases where aging is important parameter. However, ex vivo microglia cannot be kept in culture for prolonged periods of time. Therefore, while this protocol extends the life of primary microglia in culture, it should be noted that the microglia behave differently from adult microglia and in vitro studies should be carefully considered when translated to an in vivo setting.

Isolating primary microglia from the cellular heterogeneity of the brain is essential to investigate their role in both physiological and pathological conditions. This protocol describes a mechanical isolation and mixed cell culture technique that provides high yield and high purity, viable primary microglial cells for in vitro study and downstream applications.

El aislamiento de la microglía Primaria cultivos mixtos de células gliales del tejido neonatal cerebro de rata

Microglia account for approximately 12% of the  total cellular population in the mammalian brain. While neurons and astrocytes are considered  the major ...

piggyBac Transposon System Modification of Primary Human T Cells

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most commonly utilizing two plasmids with one expressing the piggyBac transposase enzyme and a transposon plasmid harboring the gene(s) of interest between inverted repeat elements which are required for gene transfer activity. PiggyBac mediates gene transfer through a "cut and paste" mechanism whereby the transposase integrates the transposon segment into the genome of the target cell(s) of interest. PiggyBac has demonstrated efficient gene delivery activity in a wide variety of insect1,2, mammalian3-5, and human cells6 including primary human T cells7,8. Recently, a hyperactive piggyBac transposase was generated improving gene transfer efficiency9,10.
Human T lymphocytes are of clinical interest for adoptive immunotherapy of cancer11. Of note, the first clinical trial involving transposon modification of human T cells using the Sleeping beauty transposon system has been approved12. We have previously evaluated the utility of piggyBac as a non-viral methodology for genetic modification of human T cells. We found piggyBac to be efficient in genetic modification of human T cells with a reporter gene and a non-immunogenic inducible suicide gene7. Analysis of genomic integration sites revealed a lack of preference for integration into or near known proto-oncogenes13. We used piggyBac to gene-modify cytotoxic T lymphocytes to carry a chimeric antigen receptor directed against the tumor antigen HER2, and found that gene-modified T cells mediated targeted killing of HER2-positive tumor cells in vitro and in vivo in an orthotopic mouse model14. We have also used piggyBac to generate human T cells resistant to rapamycin, which should be useful in cancer therapies where rapamycin is utilized15.
Herein, we describe a method for using piggyBac to genetically modify primary human T cells. This includes isolation of peripheral blood mononuclear cells (PBMCs) from human blood followed by culture, gene modification, and activation of T cells. For the purpose of this report, T cells were modified with a reporter gene (eGFP) for analysis and quantification of gene expression by flow cytometry.
PiggyBac can be used to modify human T cells with a variety of genes of interest. Although we have used piggyBac to direct T cells to tumor antigens14, we have also used piggyBac to add an inducible safety switch in order to eliminate gene modified cells if needed7. The large cargo capacity of piggyBac has also enabled gene transfer of a large rapamycin resistant mTOR molecule (15 kb)15. Therefore, we present a non-viral methodology for stable gene-modification of primary human T cells for a wide variety of purposes.

piggyBac Transposon System Modification of Primary Human T Cells

We describe a method to genetically modify primary human T cells with a transgene using the non-viral piggyBac transposon system. T cells modified to using the piggyBac transposon system exhibit stable transgene expression.

piggyBac Transposón modificación del sistema de células T humanas primarias

The piggyBac transposon system is naturally active, originally derived from the cabbage looper moth1,2. This non-viral system is plasmid based, most ...

Generation of Human Alloantigen-specific T Cells from Peripheral Blood

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide antigens presented by MHC (human ortholog HLA) molecules through the T cell receptor (TCR) in a highly sensitive and specific manner. While the primary function of T cells is to mediate protective immune responses to foreign antigens presented by self-MHC, T cells respond robustly to antigenic differences in allogeneic tissues. T cell responses to alloantigens can be described as either direct or indirect alloreactivity. In alloreactivity, the T cell responds through highly specific recognition of both the presented peptide and the MHC molecule. The robust oligoclonal response of T cells to allogeneic stimulation reflects the large number of potentially stimulatory alloantigens present in allogeneic tissues. While the breadth of alloreactive T cell responses is an important factor in initiating and mediating the pathology associated with biologically-relevant alloreactive responses such as graft versus host disease and allograft rejection, it can preclude analysis of T cell responses to allogeneic ligands. To this end, this protocol describes a method for generating alloreactive T cells from naive human peripheral blood leukocytes (PBL) that respond to known peptide-MHC (pMHC) alloantigens. The protocol applies pMHC multimer labeling, magnetic bead enrichment and flow cytometry to single cell in vitro culture methods for the generation of alloantigen-specific T cell clones. This enables studies of the biochemistry and function of T cells responding to allogeneic stimulation.

This article describes a method for the generation and propagation of human T cell clones that specifically respond to a defined alloantigen. This protocol can be adapted for cloning human T cells specific for a variety of peptide-MHC ligands.

Generación de células T humanas aloantígeno-específicos de la sangre periférica

The study of human T lymphocyte biology often involves examination of responses to activating ligands. T cells recognize and respond to processed peptide ...

Generation of Recombinant Human IgG Monoclonal Antibodies from Immortalized Sorted B Cells

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, including the development of immunotherapeutics. However, the techniques to identify and produce such antibodies tend to be arduous and sometimes the heavy and light chain pair of the antibodies are dissociated. Here, we describe a relatively simple, straightforward protocol to produce human recombinant monoclonal antibodies from human peripheral blood mononuclear cells using immortalization with Epstein-Barr Virus (EBV) and Toll-like receptor 9 activation. With an adequate staining, B cells producing antibodies can be isolated for subsequent immortalization and clonal expansion. The antibody transcripts produced by the immortalized B cell clones can be amplified by PCR, sequenced as corresponding heavy and light chain pairs and cloned into immunoglobulin expression vectors. The antibodies obtained with this technique can be powerful tools to study relevant human immune responses, including autoimmunity, and create the basis for new therapeutics.

Synthesis of human monoclonal antibodies is the first step in studies aimed at unraveling the pathophysiological mechanisms of auto-antibody-mediated immune responses. We have developed a protocol to generate recombinant human immunoglobulin G (IgG) monoclonal antibodies from blood sorted B cells, including B-cell isolation, antibody cloning and in vitro synthesis.

Generación de recombinante humano IgG anticuerpos monoclonales a partir de células inmortalizadas B Ordenada

Finding new methods for generating human monoclonal antibodies is an active research field that is important for both basic and applied sciences, ...

Preparation and Applications of Organotypic Thymic Slice Cultures

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Preparación y aplicaciones de Organotípicos tímico Slice culturas

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell ...

Generation of Human Monocyte-derived Dendritic Cells from Whole Blood

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition receptors (PRRs) expressed on their cell surface and thereby activate and regulate immunity. 
The major function of DCs is the induction of adaptive immunity in the lymph nodes by presenting antigens via MHC I and MHC II molecules to na&#239;ve T lymphocytes. Therefore, DCs have to migrate from the periphery to the lymph nodes after the recognition of pathogens at the sites of infection. For in vitro experiments or DC vaccination strategies, monocyte-derived DCs are routinely used. These cells show similarities in physiology, morphology, and function to conventional myeloid dendritic cells. They are generated by interleukin 4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF) stimulation of monocytes isolated from healthy donors. Here, we demonstrate how monocytes are isolated and stimulated from anti-coagulated human blood after peripheral blood mononuclear cell (PBMC) enrichment by density gradient centrifugation. Human monocytes are differentiated into immature DCs and are ready for experimental procedures in a non-clinical setting after 5 days of incubation.

Here, we demonstrate how monocytes are isolated by magnetic bead separation from peripheral blood mononuclear cells after density gradient centrifugation of human anti-coagulated blood. Following incubation for 5 days, human monocytes are differentiated into immature dendritic cells and are ready for experimental procedures in a non-clinical setting.

Generación de células dendríticas derivadas de monocitos humanos de sangre entera

Dendritic cells (DCs) recognize foreign structures of different pathogens, such as viruses, bacteria, and fungi, via a variety of pattern recognition ...

Induction of an Inflammatory Response in Primary Hepatocyte Cultures from Mice

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the uremic milieu, oxidative stress, endotoxemia and the decreased clearance of circulating proinflammatory cytokines by producing a large number of acute-phase reactants. Experimental tools to study inflammation and the underlying role of hepatocytes are crucial to understand the regulation and contribution of hepatic cytokines to a systemic acute phase response and a prolonged pro-inflammatory scenario, especially in an intricate setting such as chronic kidney disease. Since studying complex mechanisms of inflammation in vivo remains challenging, resource-intensive and usually requires the usage of transgenic animals, primary isolated hepatocytes provide a robust tool to gain mechanistic insights into the hepatic acute-phase response. Since this in vitro technique features moderate costs, high reproducibility and common technical knowledge, primary isolated hepatocytes can also be easily used as a screening approach. Here, we describe an enzymatic-based method to isolate primary murine hepatocytes, and we describe the assessment of an inflammatory response in these cells using ELISA and quantitative real-time PCR.

Here, we show an enzymatic approach to isolate primary hepatocytes from adult mice, and we describe the quantification of an inflammatory response using ELISA and real-time PCR.

La inducción de una respuesta inflamatoria en la Primaria hepatocitos culturas de ratones

The liver plays a decisive role in the regulation of systemic inflammation. In chronic kidney disease in particular, the liver reacts in response to the ...

Small RNA Transfection in Primary Human Th17 Cells by Next Generation Electroporation

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated from na&#239;ve CD4+ T cells in vitro by activating them in the presence of the polarizing cytokines IL-1&#946;, IL-6, IL-23, and TGF&#946;. Th17 cells orchestrate immunity against extracellular bacteria and fungi, but their aberrant activity has also been associated with several autoimmune and inflammatory diseases. Th17 cells are identified by the chemokine receptor CCR6 and defined by their master transcription factor, ROR&#947;t, and characteristic effector cytokine, IL-17A. Optimized culture conditions for Th17 cell differentiation facilitate mechanistic studies of human T cell biology in a controlled environment. They also provide a setting for studying the importance of specific genes and gene expression programs through RNA interference or the introduction of microRNA (miRNA) mimics or inhibitors. This protocol provides an easy to use, reproducible, and highly efficient method for transient transfection of differentiating primary human Th17 cells with small RNAs using a next generation electroporation device.

Next generation electroporation is an efficient method for transfecting human Th17 cells with small RNAs to alter gene expression and cell behavior.

Small RNA Transfección en células primarias Th17 humano por Next Generation electroporación

CD4+ T cells can differentiate into several subsets of effector T helper cells depending on the surrounding cytokine milieu. Th17 cells can be generated ...