Os autores gostariam de agradecer a Sally Roberts (University of Birmingham, Birmingham Reino Unido) para a dom tipo do anticorpo E1E4. Este trabalho foi financiado por uma concessão do Instituto Nacional do Câncer (4R00CA137160-03).

1. Preparação para culturas Jangada organotípicas <ol><li> As grelhas de jangada de metal muito primeiro ser tratados com ácido sulfúrico crómico para remover qualquer resíduo que poderia interferir com o processo de diferenciação. Grelhas de metal imergir em um copo de vidro contendo ácido sulfúrico durante uma hora, em seguida, lavado continuamente durante a noite com água da torneira. Após o enxaguamento durante a noite, as grades de jangada deve ser lavado durante 3-5 horas em água destilada duas vezes. </li><li> Para fornecer suporte para as jangadas, dobre três lados da jangada cerca de 0,5 cm a igual distância uns dos outros. As grelhas de metal deve então ser autoclavados. </li><li> Preparar o tampão de reconstituição 10X por adição de 2,2 g de NAHCO3 e 4,8 g de Hepes. Dissolver em 100 ml de 0,05 M de NaOH. Filtrar esterilizar e armazenar em alíquotas de 5 ml a -20 ° C. </li><li> Preparar o DMEM 10X sem bicarbonato de sódio. Filtrar esterilizar e armazenar em alíquotas de 5 ml a -20 ° C. </li><li> Preparar factor de crescimento epidérmico do rato: Dissolver 100 mg de EGF e 10 mg de BSA cada um em 10 mL de água desionizada DDQ 2 0. Combinar EGF e BSA e adicionar a 80 ml ​​de desionizada DDQ 2 0 para um volume total de 100 ml. Filtre-esterilizar alíquota, (5 ml) e guarde na -20 ° C. Para cada litro de meio E adicionar 5 ml de 1 mg / ml EGF (concentração final de 5 ng / ml). </li></ol> 2. Preparação de géis de colágeno <ol><li> Um gel de colágeno é necessário para cada balsa. O colagénio deve ser mantido em gelo para evitar que ele solidificação. Cada gel de colagénio requer 3 ml de mistura de colagénio que consiste em: 2,4 ml de colagénio do tipo frio Rat cauda-de-I (concentração final de 3-4 mg / ml), 0,3 ml de tampão de reconstituição 10X, 0,3 ml de DMEM 10X e1-2 do rato X 10 6 3T3 J2 fibroblastos como células alimentadoras. Para soluções de concentração elevada de colagénio da cauda de rato, diluir em 0,02 M de ácido acético para se atingir a concentração correcta de trabalho. </li><li> Determinar o número de géis de colágeno (balsas), você vai precisar e, em seguida, calcular um master mix para a quantidade de colágeno, reconstit 10Xtampão ution, DMEM 10X e J2 fibroblastos necessário. Incluem o suficiente em cada mistura de mestre para 3-4 géis extra devido à viscosidade do colagénio. </li><li> Para preparar o gel de colágeno, trypsinize os J2 fibroblastos e neutralizar com o meio. Misture todos os fibroblastos e coloque em um frasco de 50 ml cônico. Contar as células para determinar o número de jangadas que podem ser feitas. Mais uma vez, 1-2x10 6 fibroblastos são obrigados por gel de colágeno e, assim, por balsa. Gire para baixo as células em baixa velocidade. </li><li> Aspirar o meio a partir do fibroblasto sedimento e ressuspender na quantidade apropriada de tampão de reconstituição 10X, 10X e DMEM colagénio para o número de jangadas calculados. Adicionar o colagénio última para evitar que o gel de solidificação muito rapidamente. Uma vez que o colagénio é adicionado, misturar rapidamente, mas com cuidado para evitar a introdução de bolhas no gel. O gel deve ser de uma cor laranja avermelhada, indicativo de que o pH correcto. Se a mistura de gel é muito amarelo, adicionar um par de gotas de filtro esterilizado 1NNaOH e misturar suavemente para se obter a cor correcta. </li><li> Adicionar 3 ml de colagénio: mistura de fibroblastos para os poços de uma placa de cultura de 6 poços. A mistura de colágeno deve ser pipetado lentamente para o lado de cada poço para evitar a geração de bolhas. Colocar a placa em um 37 ° C incubador de cultura de tecidos e deixa-se solidificar durante 30 minutos. </li><li> Após 30 minutos, adicionar 3 ml de meio de E com EGF para o topo do gel de colagénio solidificado e retornar a placa para a incubadora. Os géis deve ser mantida a 37 ° C durante pelo menos um dia, mas utilizadas dentro de quatro dias. Resultados óptimos ocorrer após dois dias de incubação. </li></ol> 3. Os queratinócitos Preparando para Diferenciação <ol><li> Queratinócitos baixa passagem deve ser aumentado para cerca de 70% de confluência. Cada balsa exige 1-2x10 6 queratinócitos. Trypsinize os queratinócitos e neutralizar com meio E com EGF. Contar as células para determinar o número de jangadas que podem ser feitas. Gire para baixo as células a SPE baixoed. Ressuspender o sedimento em 1 ml de meio de E com EGF para cada cultura jangada calculado. </li><li> Aspirar o meio a partir dos géis de colagénio por da inclinação da placa de 6 poços. Adicionar 2 ml de meio fresco E com EGF para cada gel. Para cada gel, cuidadosamente adicionar 1 ml de queratinócitos ressuspensos para a 2 ml de meio E já presente no poço. Gentilmente pipeta os queratinócitos mais a 2 ml de mídia e, em seguida, deixar para trás queda gota a gota, para o gel. Agitação ou agitando a placa pode resultar na distribuição desigual de células para um lado do gel de colagénio. Colocar a placa na incubadora a 37 ° C. </li><li> As células devem ser cultivadas até à confluência, substituindo o meio E por dia. As células são confluentes quando as alterações médias para um dia amarelo após uma mudança de meio. Isso normalmente leva 2-4 dias. Se o meio não mudou por quatro dias, proceda para a próxima etapa. </li></ol> 4. Fazendo as culturas Jangada <ol><li> Use uma pinça estéril, colocar uma grade jangada estéril metal, dobrados para baixo os lados, Em um prato de 10 cm. Aspirar o meio a partir de gel de colagénio. Para soltar o gel a partir dos lados do poço, a volta do perímetro do gel com uma espátula estéril usando um movimento para cima e para baixo. Para remover o gel de colagénio, incline a placa e levante colocando um debaixo espátula. Colocar o gel de colagénio na grelha de metal sem a geração de bolhas entre a grade eo gel. Dois géis podem ser colocadas em cada grade. </li><li> A fim de fazer uma interface ar-líquido, adicionar meio E sem EGF lentamente para o fundo do prato de modo a grade jangada está a tocar os meios de comunicação, mas o gel de colagénio não é. A mídia não deve vir através dos buracos na grade. Adicionar lentamente o meio para evitar a geração de bolhas sob a grade, como isso vai impedir a diferenciação uniforme. Incubar as jangadas a 37 ° C e alterar o meio de todos os outros dias mantendo a interface ar-líquido. Colha as balsas depois de 14 dias. </li></ol> 5. Os resultados representativos Quando tele protocolo é efectuada correctamente com queratinócitos primários, este procedimento irá resultar em um epitélio bem diferenciado onde as diferentes camadas da pele são evidentes. Este ser alcançada através de exame histológico pela coloração seções de culturas de jangada para hematoxilina e eosina (H &amp; E), como mostrado na Figura 1B. HE também é útil para examinar a morfologia de jangadas eo efeito de vírus ou de compostos antivirais sobre a capacidade das células para a estratificação e diferenciar. Por exemplo, jangadas feitas a partir de HPV-imortalizadas primárias de queratinócitos humanos (HFK-31) são notavelmente mais espessa em comparação com jangadas feitas a partir de queratinócitos normais de prepúcio humano (HFK), reflectindo um aumento da taxa de proliferação ea capacidade das proteínas de HPV para manter as células de diferenciação activa no ciclo celular (Figura 1B) 10. Isto resulta em retenção dos núcleos ao longo do epitélio, enquanto que os queratinócitos normais sair do ciclo celular na diferenciação, resultando em um colapso doenvelope nuclear. Para examinar mais de perto a diferenciação, imuno-histoquímica pode ser realizada para examinar marcadores de diferenciação específicos, incluindo citoqueratinas, bem como involucrina, e filagrina. Citoqueratinas diferentes são expressos em fases específicas de diferenciação 7. Como mostrado na Figura 2, citoqueratina 10 (K10) não é expresso na camada basal, e é encontrado apenas nas camadas suprabasais que consistem em células progressivamente de diferenciação. Em jangadas de HPV positivos, a expressão de K10 é atrasado em comparação com a de queratinócitos normais, novamente reflectindo a influência de proteínas de HPV na diferenciação de queratinócitos. A imuno-histoquímica também pode ser usado para examinar o perfil de expressão de genes virais. Mostrada na Figura 3 é uma coloração representante examinar a expressão da proteína de HPV E1 ^ E4, que é uma proteína tardia cuja expressão é restrita às camadas superiores do epitélio. Como esperado, sem coloração é observada em culturas de jangada de Huma normaisn queratinócitos prepúcio. <img alt="A Figura 1" src="/files/ftp_upload/3668/3668fig1.jpg" /> Figura 1. Um esboço) de método para a preparação de culturas organotípicas de jangada de queratinócitos primários ou linhas celulares epiteliais. B) coloração de hematoxilina e eosina de culturas organotípicas de jangada gerados a partir de queratinócitos do prepúcio humano de forma estável mantendo HPV-31 (HFK-31) episomes, ou a partir normais queratinócitos do prepúcio humano (HFK). As camadas individuais epiteliais são identificados, bem como a ficha de colagénio. <img alt="A Figura 2" src="/files/ftp_upload/3668/3668fig2.jpg" /> Figura 2. Citoqueratina 10 expressão (K10) é limitada às camadas suprabasais do epitélio. A imuno-histoquímica foi realizada em secções transversais de culturas organotípicas de jangada gerados a partir de HFK-31 células, bem como HFKs normais utilizando um anticorpo para K10. DNA celular foi contrastadas com DAPI. As imagens foram capturadas confmicroscopia de fluorescência Ocal. As setas indicam a camada basal do epitélio. <img alt="A Figura 3" src="/files/ftp_upload/3668/3668fig3.jpg" /> Figura 3. A E1 HPV ^ proteína E4 é produzido no final da fase produtiva do ciclo de vida virai. A imuno-histoquímica foi realizada em secções transversais de culturas organotípicas de jangada gerados a partir de HFK-31 células, bem como HFKs normais utilizando anticorpos para E1E4. DNA celular foi contrastadas com DAPI. As imagens foram capturadas usando microscopia de fluorescência confocal. As setas indicam a camada basal do epitélio.

<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>

Não temos nada a divulgar.

Descrevemos aqui um método que pode ser usado para estudar a diferenciação epitelial em geral, mas também pode ser facilmente adaptado para estudar patogénese virai, bem como a eficácia da terapêutica potencial. Muitos vírus alvo células epiteliais, quer como o principal local de infecção, como no caso de HPV, ou em algum ponto do ciclo de vida do vírus, tal como com herpesvírus. Embora crescente culturas jangada organotípicas é demorado, a habilidade de recapitular fielmente na diferenciação e...

<table border="1"><tbody><tr><td> Nome do Reagente </td><td> Companhia </td><td> Número de Catálogo </td></tr><tr><td> Rato O colágeno tipo cauda 1 (4-5mg/ml) </td><td> BD Biosciences </td><td> 354236 </td></tr><tr><td> DMEM sem niveis de NaHC0 3 </td><td> Invitrogen </td><td> 12100-061 </td></tr><tr><td> 3 niveis de NaHC0 </td><td> Sigma </td><td> S5761 </td></tr><tr><td> Hepes </td><td> Calbiochem </td><td> 391338 </td></tr><tr><td> Grades metálicas de aço inoxidável </td><td> Williams e Mettle Co. </td><td> CR-03063-040-100-S </td></tr><tr><td> E média 11 </td><td></td><td></td></tr><tr><td> Fator de Crescimento Epidérmico </td><td> BD Biosciences </td><td> 354010 </td></tr></tbody></table>

generation of organotypic raft cultures from primary human keratinocytes

O desenvolvimento de culturas organotípicas de jangada epiteliais forneceu investigadores com um eficiente sistema in vitro que fielmente diferenciação recapitula epitelial. Há muitos usos para esse sistema. Por exemplo, a capacidade de crescer tridimensionais culturas organotípicas de jangada de queratinócitos foi um passo importante no estudo do papilomavírus humano (HPV) 1. O ciclo de vida de HPV é rigidamente ligada à diferenciação de epitélio escamoso 2. Organotípicas culturas jangada epiteliais como demonstrado aqui reproduzir o ciclo de vida papilomavírus, incluindo a produção de vírus 3,4,5. Além disso, estas culturas jangada exibem lesões displásicas semelhantes aos observados após infecção in vivo com HPV. Assim, o presente sistema pode também ser usado para estudar cancros de células epiteliais, bem como o efeito de drogas na diferenciação de células epiteliais em geral. Originalmente desenvolvido pela Asselineau e Prunieras <sup&gt; 6 e modificado por Kopan et al. 7, o sistema de cultura organotípico epitelial jangada amadureceu em geral um modelo de cultura, relativamente fácil, o qual envolve o crescimento de células em colagénio liga mantida a uma interface ar-líquido (Figura 1A). Ao longo de 10-14 dias, as células estratificar e diferenciar, formando um epitélio espessura total que produz-diferenciação específicos citoqueratinas. Jangadas colhidas pode ser examinado histologicamente, bem como por técnicas padrão de moleculares e bioquímicos. Neste artigo, é descrito um método para a geração de culturas primárias de jangada de queratinócitos humanos. A mesma técnica pode ser usada com estabelecidas linhas de células epiteliais, e pode facilmente ser adaptado para uso com o tecido epitelial a partir de biópsias normais ou doentes 8. Muitos vírus alvo tanto o epitélio cutâneo ou da mucosa, como parte de seu ciclo de vida replicativo. Ao longo dos últimos anos, a viabilidade do uso culto jangada organotípicoUres como um método de estudo das interacções vírus-hospedeiro de células tem sido demonstrado para herpesvírus vários, bem como adenovírus, parvovírus, e poxvírus 9. Culturas organotípicas de jangada pode assim ser adaptado para examinar patogénese virai, e são os únicos meios para testar novos agentes antivirais para aqueles vírus que não são cultiváveis ​​em linhas de células 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

Um<em&gt; In vitro</em&gt; Método para simular<em&gt; In vivo</em&gt; Diferenciação epitelial é descrito. Muitos vírus alvo células epiteliais, como parte do seu ciclo de vida do vírus, e este método proporciona um meio de análise de vírus: interacções hospedeiras que mais se assemelha a que ocorre<em&gt; In vivo</em&gt;. Esta técnica pode ser usada com queratinócitos primários, linhas celulares estabelecidas, bem como normais ou doentes de tecidos de biópsia.

Geração de Culturas Raft organotípicas de queratinócitos humanos primários

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 visualizações.  University of North Carolina-Chapel Hill. Um In vitro Método para simular In vivo Diferenciação epitelial é descrito. Muitos vírus alvo células epiteliais, como parte do seu ciclo de vida do vírus, e este método proporciona um meio de análise de vírus: interacções hospedeiras que mais se assemelha a que ocorre In vivo. Esta técnica pode ser usada com queratinócitos primários, linhas celulares estabelecidas, bem como normais ou doentes de tecidos de bióp...

Vídeo: 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.

Um método simples e eficiente para isolar Macrófagos de Mixed culturas primárias de células do fígado Adulto

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

Imunologia e Infecção

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.

Geração de Células T reguladoras induzidas a partir de células T humanas primárias Naïve e Memória

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.

Isolamento Microglia primária a partir de cultura mista de células gliais de tecido de cérebro de rato neonatal

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 modificação do sistema Transposon de células T primárias Humanos

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.

Geração de Células T humanas alo-específicos a partir de Sangue Periférico

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.

Geração de IgG Humana Recombinante de Anticorpos Monoclonais As células seleccionadas a partir de B imortalizadas

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.

Preparação e Aplicações de culturas organotípicas Tímico Fatia

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.

Geração de células dendríticas derivadas de monócitos humanos a partir de sangue inteiro

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.

A indução de uma resposta inflamatória em culturas primárias de hepatócitos de ratinhos

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.

RNA pequeno Transfecção em células primárias Th17 humano por Next Generation electroporação

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