Agradecemos a Agência de Recuperação Vida Alliance Organ, da Universidade de Miami para fornecer pulmões. Agradecemos também o Dr. Lisa Künzi para a preparação de DNA utilizado para os experimentos mostrados na figura 2B e 3-D, Dr. Ben Gerovac e Lisa Novak para o vírus mCherry utilizado para os experimentos nas Figuras 3A a C. Também agradecemos Gabriel Gaidosh a partir da instalação de imagem do Departamento de Oftalmologia da Universidade de Miami. Este estudo foi patrocinado por doações do NIH, a Fundação CF eo hospedeiros de bordo Medical Research Institute com o Dr. Matthias Salathe.

1. Fase 1: Cultura da HEK 293T <ol><li> Prepare meios células HEK (referido como meio de HEK) por adição de 10% (v / v) de soro fetal de bovino (FBS) e 1% (v / v) de penicilina / estreptomicina a elevada glicose de Eagle Modificado Meio de Dulbecco (DMEM). </li><li> Bata de um prato de 10 cm de cultura de tecidos com colagénio I. Mistura de 30 ul de colagénio I, em 2 ml de água destilada. Espalhar a mistura sobre o prato e incubar durante 2 horas a 37 ° C e 5% de CO 2. </li><li> Remover a mistura e deixar secar o prato numa câmara de fluxo laminar durante 15 min. </li><li> Placa células HEK para a placa revestida a uma densidade de 1 x 10 6 células em 10 ml de meio de HEK e incubar a 37 ° C e 5% de CO 2. </li><li> Quando as células HEK são 50-60% confluentes (Figura 1a, 2-3 dias após o plaqueamento), prossiga para a fase 2. Visual avaliação da confluência é suficiente. Sem quantificação é necessária. Nota: as células HEK são cultivadas em meios de HEK (ver acima). Quando eles atingem a confluência, tEi pode ser dividido (por exemplo, 1/5) para baixo ou congelado para uma utilização posterior. </li></ol> 2. Fase 2: Produção e Concentração dos lentivírus (VEs) em células HEK Nota: Os protocolos devem ser executadas em conformidade com as normas institucionais e governamentais sob + condições (Enhanced BSL-2) nos EUA BSL-2. <ol><li> Transfectar utilizando um procedimento de precipitação de cálcio (lipofectamina e outros agentes de transfecção não fosse tão eficiente quanto o procedimento de precipitação de cálcio). O uso de um kit comercial de transfecção é recomendado para assegurar a reprodutibilidade, embora o processo é modificado como se segue. Trazer todos os reagentes à temperatura ambiente. Este é o dia 0. </li><li> Para cada placa de 10 cm de células HEK293, misturar 4,5 g de envelope de ADN PMD2-VSVG, 7,5 ug de embalagem ADN pMDLg / pRRE, 3,75? G de ADN de pRSV-Rev, 10 ug de ADN do vector de expressão de lentivírus, 86 ul de uma solução 2 M de cálcio , e água estéril num tubo de centrífuga de 15 ml para um volume final de 700 ul(As duas últimas soluções são fornecidas no kit de transfecção comercial). </li><li> Gota a gota, adicionar 700 ul de solução salina tamponada com HEPES 2x (HBS, fornecidas com o kit de transfecção) aquando da mistura (na câmara de fluxo laminar) e incubar a temperatura ambiente durante 30 min (efectuar o passo 4, enquanto espera). </li><li> Durante o tempo de incubação inicial, depósito de 5 ul da mistura sobre uma lâmina. Verifique a gota sob um microscópio com uma objetiva de 10X, concentrando-se lentamente para cima e para baixo. Um precipitado fino deve ser visível, confirmando o processo de precipitação de fosfato de cálcio (Figura 1b). </li><li> Pare a precipitação após 30 minutos por adição de 10 ml de células HEK media e pipeta cima e para baixo para misturar. </li><li> Leve o prato de células HEK do passo 2,1-2,5, e remover o meio (dia 0). Adicionar a solução do passo 2.5 precipitado cuidadosamente e lentamente ao longo da borda do prato. </li><li> No dia seguinte (dia 1), remova e descarte o meio contendo o precipitado e substituí-lo wom 10 ml de meio de HEK. Verifique se o precipitado fino sob o microscópio: deve ser visível no prato em espaços vazios entre as células (Figuras 1C e D). </li><li> No dia 2, recolhe meios de comunicação com uma seringa, filtrar através de um filtro de seringa de 0,45 um para um tubo de centrífuga de 15 ml contendo 3,8 ml de um polietileno-glicol 40% (PEG). Inverter 5x para misturar e armazenar a 4 ° C durante pelo menos 24 horas (para precipitar o PEG para formar, mas não mais do que 5 dias) até que todos os conjuntos de vírus estão completas. </li><li> Repita o passo 2.8 nos dias 3 e 4. No dia 4 não coloque meio HEK mas descontaminar com 10% de água sanitária e rejeitar a placa. </li><li> Centrífuga de todos os tubos de colheita (geralmente todas as coleções em conjunto no dia 5) a 1.650 g durante 20 min a 4 ° C para sedimentar o vírus. Retirar e descartar o sobrenadante, e girar novamente a 1.650 xg por 5 min a 4 ° C para recolher e remover qualquer remanescente de PEG sobrenadante. </li><li> Resuspend a pelete de cada tubo, em 200 ul de meio de crescimento epitelial brônquica (BEGM) sem anfotericina B 8. Reuni-los em conjunto e a aliquota de 200 ul. vírus armazenar a -80 ° C. Alíquota um adicional de 10 ul de realizar o ensaio ELISA Gag de HIV-1 (p24) antigénio. </li><li> Utilizando um kit de ELISA comercial, realizar um ensaio de antígeno p24 de acordo com o protocolo do fabricante. O ensaio dá uma estimativa da p24 viral em pg / ml. </li></ol> 3. Fase 3: células de cultura primária HBE <ol><li> Placa HBE células em 10 ml BEGM meios (100 ul com anfotericina B se 0 passagem células são usados ​​para eliminar a possibilidade de contaminação por fungos) a uma densidade de 1 x 10 6 células em colagénio I revestido 10 centímetros prato de cultura de tecidos. Incubar a 37 ° C e 5% de CO 2. </li><li> No dia seguinte, remover o meio e substituir por 10 ml BEGM (com 100 ul de anfotericina B por células passagem 0) para um total de 4 dias. Voltar para incubadora. </li><li> Após 4 days, use apenas BEGM sem meios anfotericina B. Alterar a cada dois dias. </li><li> Quando as células são 80-90% confluentes (Figura 2a; ~ 7 dias após o plaqueamento), proceda com a transdução (preste atenção a 3,5 para preparação antes de transdução). Novamente, a avaliação visual de confluência é suficiente. Sem quantificação é necessária. </li><li> Antes de transdução, revestir os insertos de suporte permeável com uma solução de colagénio IV diluídos a 1/10 com água destilada estéril. Adicionar 200 mL de cada inserção e deixar secar durante a noite na câmara de fluxo laminar. </li></ol> 4. Etapa 4: Transdução de Células HBE <ol><li> Remover o meio, adicionar 3 ml de tripsina a 0,1% em ácido etilenodiaminotetracético 1 mM em salino tamponado com fosfato (EDTA / PBS) e incuba-se 5 minutos a 37 ° C e 5% de CO 2. Verifique sob o microscópio (objetiva de 10X) para ver se todas as células são separadas. Se não, voltar para a incubadora para um adicional de 5 min. </li><li> Quando todas as células são separadas, adicione 3ml de inibidor de tripsina de soja (SBTI 1 mg / ml), misturar e transferir para um tubo de centrífuga de 15 ml. Agregar as células por centrifugação a 460 xg durante 5 min à temperatura ambiente. </li><li> Remover o sobrenadante e pellet ressuspender em 3 ml de BEGM e prosseguir com a contagem. </li><li> Ressuspender as células (HBE agora uma passagem ou P1) a uma razão de 2 x 10 5 células por um suporte permeável a inserção de 12 mm de 400 ul de BEGM (ver Tabela 2). Extrapolar estes números com base na quantidade de pastilhas de suporte permeável que vai ser transduzidas. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> Superfície </td><td> área de superfície </td><td> A contagem de células </td><td> meios de comunicação </td><td> Volume (media) </td></tr><tr><td> Placa de 10 cm </td><td> 52,7 cm 2 </td><td> 1 x 10 6 </td><td> BEGM </td><td> 10 ml </td> &lt;/ Tr&gt; <tr><td> filtro de 12 milímetros </td><td> 1,13 cm 2 </td><td> 2 x 10 5 </td><td> BEGM </td><td> 400 ul </td></tr><tr><td> filtro de 24 milímetros </td><td> 4.52 cm 2 </td><td> 8 x 10 5 </td><td> BEGM </td><td> 800 ul </td></tr></tbody></table> Tabela 2: Meios extrapolações por contagem de células. <ol start="5"><li> Usando uma multiplicidade de infecção fator (MOI) de 4, adicionar o volume apropriado de vírus à suspensão de células. </li><li> Adiciona-se brometo de hexadimetrina a um / ml de concentração final de 2 ug. </li><li> Dispense 400 ul da mistura (BEGM, HBE células, vírus e brometo Hexadimetrina) por inserção de suporte permeável no compartimento apical, e adicionar 1 ml de BEGM sozinho para o compartimento basolateral. Incubar durante a noite a 37 ° C e 5% CO 2. Células Sempre placa HBE sem vírus como controle e seleção de teste puromicina se for o caso. </li><li> No dia seguinte, REMmídia apical e basolateral ove, lavar com PBS, e substituir os dois compartimentos com interface ar-líquido (ALI) media 8. </li><li> Quando as células estão confluentes, remover o líquido apical (conhecido como o estabelecimento de uma interface ar-líquido). Continuar a alterar o meio (ALI) no compartimento de fundo a cada dois dias até atingirem a maturidade plena; geralmente de 3 a 4 semanas mais tarde. Nota: Se o vector lentiviral que contém um gene de selecção tal como a puromicina, as células podem ser seleccionadas através da adição de puromicina de acordo com uma curva de concentração de selecção. Para as células HBE, uma concentração de 1 ug / ml foi determinado a ser ideal para a selecção de células transduzidas consistente. No entanto, esta concentração pode ser diferente para outras células ou condições e deve ser determinada por curvas de matar. A adição de puromicina pode começar tão cedo quanto um dia depois de os meios de comunicação foi substituída pela mídia ALI ou posteriores (2-3 dias) para permitir a plena expressão do gene de selecção. Certifique-se de adicionar puromicina a uma inserção que hcomo não foram transduzidas. Puromicina pode ser adicionado até que as células são totalmente diferenciadas. </li></ol>

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The authors have nothing to disclose.

O protocolo aqui descrito assegura perto de 100% de eficiência de transdução. No entanto, existem passos durante o processo, que são importantes para alcançar este objectivo. Por exemplo, durante o processo de transfecção, a presença de precipitados na mistura de transdução (gota) ou no prato do dia após a transdução (Figuras 1b e d) são ambos relevantes para uma boa transfecção. A ausência de um precipitado ou a presença de aglomerados pode ser devido a uma omissão d...

O desenvolvimento de lentivírus, pseudotipados replicação deficiente (LV) forneceu aos investigadores um método seguro e eficiente para entregar transgenes in vitro 1. Devido à sua expressão a longo prazo, estes LV também poderia ser utilizada para a entrega de agentes terapêuticos in vivo 2,3. A produção de LV requer a co-transfecção de células de rim embrionário humano (HEK) com vários plasmídeos: vector de transferência, gag / pol de empacotamento, rev embalagem, e envelope glicoproteína do vírus da estomatite vesicular (VSV-G) plasmídeos. Sistemas de empacotamento de terceira geração são considerados mais seguros do que os sistemas de segunda geração, porque o gene rev é codificada num plasmídeo de empacotamento separada, reduzindo assim a possibilidade de recombinação para produzir um lentivírus competente de replicação. Embora os sistemas de embalagem de segunda geração deu cinco unidades de transdução totais mais elevados de dobra, a divisão do genoma viral original para criaro sistema de terceira geração diminuiu o nível de transdução de apenas minimamente 3,4. Enquanto linhas celulares podem ser transduzidas mais facilidade e eficiência, os investigadores mudaram seu foco para as células primárias, porque estes são mais representativos de tecidos in vivo 5,6. No entanto, as células primárias são refratários a transdução. Enquanto a transdução de células epiteliais das vias aéreas totalmente diferenciadas têm sido relatados, a eficiência global não excedeu 15% 7. Aqui descrito é um protocolo que permite a produção de LVS-de título elevado. Uma abordagem passo-a-passo é delineada para atingir quase 100% de eficiência de transdução em células HBE primários. Mais especificamente, o protocolo descreve as condições óptimas de cultura para ter sucesso 3 instrumentais. Resumidamente, as células HEK 293T são transfectadas usando o pCDH lentiviral vector de expressão com o promotor de EF-1 dirigindo a expressão de verde fluorescente melhoradaproteína (eGFP) ou mCherry, uma proteína fluorescente vermelha, e um sistema de empacotamento de terceira geração. LVs libertados para os meios de comunicação são coletadas de 24 a 72 horas mais tarde. As partículas de vírus são concentradas com polietilenoglicol (PEG), um método conveniente e fácil de concentração virai, antes da estimação título utilizando um kit de ensaio imuno antigénio p24 ligado a enzima (ELISA). Subsequentemente, as células HBE primárias indiferenciadas são transduzidas em meios de proliferação (meio de crescimento epitelial brônquica ou BEGM) 8, quando a montar (depois de ser tripsinizadas), a uma multiplicidade de infecção factor 4 (MOI), a adição de brometo de hexadimetrina e incubando durante 16 horas ( pernoite). As células são então deixadas diferenciar. Uma vez que o transgene é expresso viral a longo prazo (usando o promotor apropriado, ver discussão), expressão vai ser mantida durante toda a diferenciação em um epitélio das vias respiratórias pseudo ou pode ser induzida (utilizando um promotor indutível) após a diferenciação. <p class = &quot;jove_content&quot;&gt; Este protocolo é de grande interesse para pesquisadores que desejam usar células HBE primários em vez de linhas celulares, e pode ser adaptável a outros difíceis de transdução de tipos de células.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>HEK293T/17 cells</td>
			<td>ATCC</td>
			<td>CRL-11268</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum (FBS)&#160;</td>
			<td>Sigma</td>
			<td>12306C</td>
			<td>use at 10%</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>Thermo Scientific</td>
			<td>11995-040</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100x)</td>
			<td>Thermo Scientific</td>
			<td>15140-122</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Collagen I</td>
			<td>BD Biosciences</td>
			<td>354231</td>
			<td>dilute 1:75 in distilled water</td>
		</tr>
		<tr>
			<td>Calphos</td>
			<td>Clontech</td>
			<td>631312</td>
			<td>Transfection kit containing 2M Calcium solution, 2X HEPES-Buffered solution (HBS) and sterile H2O</td>
		</tr>
		<tr>
			<td>Polythylene glycol 8000</td>
			<td>VWR scientific</td>
			<td>101108-210</td>
			<td>stock of 40%</td>
		</tr>
		<tr>
			<td>Amphotericin B</td>
			<td>Sigma</td>
			<td>A9528</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Trypsin</td>
			<td>Sigma</td>
			<td>T4799</td>
			<td></td>
		</tr>
		<tr>
			<td>Soybean trypsin inhibitor</td>
			<td>Sigma</td>
			<td>T9128</td>
			<td></td>
		</tr>
		<tr>
			<td>Transwell 12mm</td>
			<td>Corning</td>
			<td>3460</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagen IV</td>
			<td>Sigma</td>
			<td>C7521</td>
			<td>dilute 1:10 in distilled water</td>
		</tr>
		<tr>
			<td>Hexadimethrine bromide</td>
			<td>Sigma</td>
			<td>H9268</td>
			<td>stock of 2mg/ml</td>
		</tr>
		<tr>
			<td>Puromycin (10.000x)</td>
			<td>Thermo Scientific</td>
			<td>A11138-03</td>
			<td>use at 1x</td>
		</tr>
		<tr>
			<td>Alliance HIV-1 p24 ELISA</td>
			<td>PerkinElmer</td>
			<td>NEK050001KT</td>
			<td></td>
		</tr>
		<tr>
			<td>F12</td>
			<td>Thermo Scientific</td>
			<td>11765-054</td>
			<td></td>
		</tr>
		<tr>
			<td>pCDH-EF1-MCS-IRES-Puro</td>
			<td>System Biosciences</td>
			<td>CD532A-2</td>
			<td>lentiviral expression vector</td>
		</tr>
		<tr>
			<td>pMD2-VSVG</td>
			<td>Addgene</td>
			<td>12259</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pMDLg/pRRE</td>
			<td>Addgene</td>
			<td>12251</td>
			<td>packaging DNA</td>
		</tr>
		<tr>
			<td>pRSV-rev</td>
			<td>Addgene</td>
			<td>12253</td>
			<td>packaging DNA</td>
		</tr>
	</tbody>
</table>

optimal lentivirus production and cell culture conditions necessary to successfully transduce primary human bronchial epithelial cells

A cultura in vitro de células primárias humanas epiteliais brônquicas (HBE) usando condições de interface ar-líquido fornece um modelo útil para estudar os processos de diferenciação e função das células das vias aéreas. Nos últimos anos, o uso de vectores lentivirais para a entrega do transgene tornou-se prática comum. Embora existam relatórios de transdução de células epiteliais das vias respiratórias totalmente diferenciadas com certos lentivírus digitado pseudo-não-HIV, a eficiência global de transdução é geralmente inferior a 15%. O protocolo aqui apresentado proporciona um método fiável e eficiente para a produção de lentivírus e a transdução de células epiteliais brônquicas humanas primárias. Usando células epiteliais brônquicas indiferenciadas, transdução em meio de crescimento epitelial brônquica, enquanto as células fixar, com uma multiplicidade de infecção de factor de 4 fornece eficiências perto de 100%. Este protocolo descreve, passo-a-passo, a preparação e concentração dos vectores lentivirais de título elevado e thprocesso de transdução de e. Discute-se as experiências que determinaram as condições de cultura ideais para atingir transduções altamente eficientes de células epiteliais brônquicas humanas primárias.

A Figura 1 ilustra diferentes passos chave de avaliar células e soluções durante o processo de transfecção. A Figura 1A mostra a confluência óptimo das células HEK293T antes da transfecção para a produção de vírus. É importante que as células são distribuídos uniformemente ao longo do prato. Figura 1b revela o precipitado em uma gota da mistura de transfecção utilizando brilhantes óptica de campo e uma objectiva 10X. ...

Watch this Scientific Journal Video about Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells at JoVE.com

Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Optimal Lentivírus produção e cultura celular condições para sucesso pilhas transdução primária brônquicas humanas epiteliais

In vitro culture of primary human bronchial epithelial (HBE) cells using air-liquid interface conditions provides a useful model to study the processes of ...

optimal-lentivirus-production-cell-culture-conditions-necessary-to

Research

JoVE Journal

Biology

19.1K visualizações.  University of Miami, Miller School of Medicine. Primary human bronchial epithelial cells are difficult to transduce. This protocol describes the production of lentiviruses and their concentration as well as the optimal culture conditions necessary to achieve highly efficient transductions in these cells throughout differentiation to a pseudostratified epithelium.

Vídeo: Optimal Lentivirus Production and Cell Culture Conditions Necessary to Successfully Transduce Primary Human Bronchial Epithelial Cells

Human ES cells: Starting Culture from Frozen Cells

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  approximately one (to two) million irradiated mouse embryonic fibroblast feeder cells is rinsed with HuES media to remove residual serum and cell debris, and then HuES media added and left to equilibrate in the cell culture incubator.  A frozen vial of cells from long term liquid nitrogen storage or a -80C freezer is sourced and quickly submerged in a 37C water bath for quick thawing.  Cells in freezing media are then removed from the vial and placed in a large volume of HuES media.  The large volume of HuES media facilitates removal of excess serum and DMSO, which can cause HuES human embryonic stem cells to differentiate.  Cells are gently spun out of suspension, and then re-suspended in a small volume of fresh HuES media that is then used to seed the MEF plate.  It is considered important to seed the MEF plate by gently adding the HuES cells in a drop wise fashion to evenly disperse them throughout the plate.  The newly established HuES culture plate is returned to the incubator for 48 hrs before media is replaced, then is fed every 24 hours thereafter.

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.

Células ES: Cultura A partir de células congeladas

Here we demonstrate how our lab begins a HuES human embryonic stem cell line culture from a frozen stock.  First, a one to two day old ten cm plate of  ...

Biologia

Lentivirus Production

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are silenced at the post-transcriptional state. Short hairpin RNAs, precursors to siRNA, can be expressed using lentivirus, allowing for RNAi in a variety of cell types. Lentiviruses, such as the Human Immunodeficiency Virus, are capable to infecting both dividing and non-dividing cells. We will describe a procedure which to package lentiviruses. Packaging refers to the preparation of competent virus from DNA vectors. Lentiviral vector production systems are based on a 'split' system, where the natural viral genome has been split into individual helper plasmid constructs. This splitting of the different viral elements into four separate vectors diminishes the risk of creating a replication-capable virus by adventitious recombination of the lentiviral genome. Here, a vector containing the shRNA of interest and three packaging vectors (p-VSVG, pRSV, pMDL) are transiently transfected into human 293 cells. After at least a 48-hour incubation period, the virus containing supernatant is harvested and concentrated. Finally, virus titer is determined by reporter (fluorescent) expression with a flow cytometer.

To make lentiviruses, DNA vectors are transfected into human 293 cells. After harvest and concentrating the supernatant, virus titer is determined by fluorescence expression with a flow cytometer.

Produção lentivírus

RNA interference (RNAi) is a system of gene silencing in living cells. In RNAi, genes homologous in sequence to short interfering RNAs (siRNA) are ...

Primary Human Bronchial Epithelial Cells Grown from Explants

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the function and structure of human epithelial cells. Here we describe in detail the method of growing bronchial epithelial cells from bronchial airway tissue that is harvested by the surgeon at the times of lung surgery (e.g. lung cancer or lung volume reduction surgery). With ethics approval and informed consent, the surgeon takes what is needed for pathology and provides us with a bronchial portion that is remote from the diseased areas. The tissue is then used as a source of explants that can be used for growing primary bronchial epithelial cells in culture. Bronchial segments about 0.5-1cm long and &le;1cm in diameter are rinsed with cold EBSS and excess parenchymal tissue is removed. Segments are cut open and minced into 2-3mm3 pieces of tissue. The pieces are used as a source of primary cells. After coating 100mm culture plates for 1-2 hr with a combination of collagen (30 &mu;g/ml), fibronectin (10 &mu;g/ml), and BSA (10 &mu;g/ml), the plates are scratched in 4-5 areas and tissue pieces are placed in the scratched areas, then culture medium (DMEM/Ham F-12 with additives) suitable for epithelial cell growth is added and plates are placed in an incubator at 37&deg;C in 5% CO2 humidified air. The culture medium is changed every 3-4 days. The epithelial cells grow from the pieces forming about 1.5 cm diameter rings in 3-4 weeks. Explants can be re-used up to 6 times by moving them into new pre-coated plates. Cells are lifted using trypsin/EDTA, pooled, counted, and re-plated in T75 Cell Bind flasks to increase their numbers. T75 flasks seeded with 2-3 million cells grow to 80% confluence in 4 weeks. Expanded primary human epithelial cells can be cultured and allowed to differentiate on air-liquid interface. Methods described here provide an abundant source of human bronchial epithelial cells from freshly isolated tissues and allow for studying these cells as models of disease and for pharmacology and toxicology screening.

Here we describe a detailed method for growing primary human bronchial epithelial cells from explants of human bronchial airway tissue including differentiated growth on an air-liquid interface. This method provides an abundant source of primary cells for investigating the role of the airway epithelium in human lung health and disease.

Primárias células epiteliais brônquicas humanas Grown a partir de explantes

Human bronchial epithelial cells are needed for cell models of disease and to investigate the effect of excipients and pharmacologic agents on the ...

Isolation and Culture of Adult Epithelial Stem Cells from Human Skin

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate daughter cells that retain the stem cell phenotype and transit-amplifying cells (TA cells) that migrate from the stem cell niche, undergo rapid proliferation and terminally differentiate to repopulate the tissue.
Epithelial stem cells have been identified in the epidermis, hair follicle, and intestine as cells with a high in vitro proliferative potential and as slow-cycling label-retaining cells in vivo 1-3. Adult, tissue-specific stem cells are responsible for the regeneration of the tissues in which they reside during normal physiologic turnover as well as during times of stress 4-5. Moreover, stem cells are generally considered to be multi-potent, possessing the capacity to give rise to multiple cell types within the tissue 6. For example, rodent hair follicle stem cells can generate epidermis, sebaceous glands, and hair follicles 7-9. We have shown that stem cells from the human hair follicle bulge region exhibit multi-potentiality 10.
Stem cells have become a valuable tool in biomedical research, due to their utility as an in vitro system for studying developmental biology, differentiation, tumorigenesis and for their possible therapeutic utility. It is likely that adult epithelial stem cells will be useful in the treatment of diseases such as ectodermal dysplasias, monilethrix, Netherton syndrome, Menkes disease, hereditary epidermolysis bullosa and alopecias 11-13. Additionally, other skin problems such as burn wounds, chronic wounds and ulcers will benefit from stem cell related therapies 14,15. Given the potential for reprogramming of adult cells into a pluripotent state (iPS cells)16,17, the readily accessible and expandable adult stem cells in human skin may provide a valuable source of cells for induction and downstream therapy for a wide range of disease including diabetes and Parkinson's disease.

A rapid, robust way of isolating viable adult epithelial stem cells from human skin is described. The method utilizes enzymatic digestion of skin collagen matrix , followed by plucking of hair follicles and isolation of single cell suspensions or tissue fragments for cell culture.

Isolamento e Cultura de Células-Tronco Adultas epitelial da pele humana

The homeostasis of all self-renewing tissues is dependent on adult stem cells. As undifferentiated stem cells undergo asymmetric divisions, they generate ...

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and uterine carcinomas, where death rates have fallen in recent years, ovarian cancer cure rates have remained relatively unchanged over the past two decades 1. This is largely due to the lack of appropriate screening tools for detection of early stage disease where surgery and chemotherapy are most effective 2, 3. As a result, most patients present with advanced stage disease and diffuse abdominal involvement. This is further complicated by the fact that ovarian cancer is a heterogeneous disease with multiple histologic subtypes 4, 5. Serous ovarian carcinoma (SOC) is the most common and aggressive subtype and the form most often associated with mutations in the BRCA genes. Current experimental models in this field involve the use of cancer cell lines and mouse models to better understand the initiating genetic events and pathogenesis of disease 6, 7. Recently, the fallopian tube has emerged as a novel site for the origin of SOC, with the fallopian tube (FT) secretory epithelial cell (FTSEC) as the proposed cell of origin 8, 9. There are currently no cell lines or culture systems available to study the FT epithelium or the FTSEC. Here we describe a novel ex vivo culture system where primary human FT epithelial cells are cultured in a manner that preserves their architecture, polarity, immunophenotype, and response to physiologic and genotoxic stressors. This ex vivo model provides a useful tool for the study of SOC, allowing a better understanding of how tumors can arise from this tissue, and the mechanisms involved in tumor initiation and progression.

Ex Vivo Culture of Primary Human Fallopian Tube Epithelial Cells

The fallopian tube (FT) is emerging as an alternative site of origin for serous ovarian carcinoma (SOC). This protocol describes a novel method for the isolation and ex vivo culture of fallopian tube epithelial cells. This system recapitulates the in vivo epithelium and allows the study of SOC pathogenesis.

Ex Vivo de Cultura Primária células epiteliais trompa de Falópio

Epithelial ovarian cancer is a leading cause of female cancer mortality in the United States. In contrast to other women-specific cancers, like breast and ...

Transduction of Human Cells with Polymer-complexed Ecotropic Lentivirus for Enhanced Biosafety

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for laboratory personnel. Pantropic lentiviruses, such as the commonly used VSV-G pseudotype, are a valuable tool for studying gene function because they can transduce many cell types, including non-dividing cells. However, researchers may wish to avoid production and centrifugation of pantropic viruses encoding oncogenes due to higher biosafety level handling requirements and safety issues. Several potent oncogenes, including c-Myc and SV40 large T antigen, are known to enhance production of induced pluripotent stem cells (iPSC). All other known iPSC-inducing genetic changes (OCT4, SOX2, KLF4, NANOG, LIN28, and p53 loss of function) also have links to cancer, making them of relatively high safety concern as well. 
While these cancer-related viruses are useful in studying cellular reprogramming and pluripotency, they must be used safely. To address these biosafety issues, we demonstrate a method for transduction of human cells with ecotropic lentivirus, with additional emphasis on reduced cost and convenient handling. We have produced ecotropic lentivirus with sufficiently high titer to transduce greater than 90% of receptor-expressing human cells exposed to the virus, validating the efficacy of this approach. 
Lentivirus is often concentrated by ultracentrifugation; however, this process takes several hours and can produce aerosols infectious to human biomedical researchers. As an alternative, viral particles can be more safely sedimented onto cells by complexation with chondroitin sulfate and polybrene (CS/PB). This technique increases the functional viral titer up to 3-fold in cells stably expressing murine retrovirus receptor, with negligible added time and cost. Transduction of human dermal fibroblasts (HDFs) is maximally enhanced using CS/PB concentrations approximately 4-fold lower than the optimal value previously reported for cancer cell lines, suggesting that polymer concentration should be titrated for the target cell type of interest. We therefore describe the use of methylthiazolyldiphenyl-tetrazolium bromide (MTT) to assay for polymer toxicity in a new cell type. We observe equivalent viability of HDFs after viral transduction using either polymer complexation or the standard dose of polybrene (PB, 6 &mu;g/ml), indicating minimal acute toxicity.
In this protocol, we describe the use of ecotropic lentivirus for overexpression of oncogenes in human cells, reducing biosafety risks and increasing the transduction rate. We also demonstrate the use of polymer complexation to enhance transduction while avoiding aerosol-forming centrifugation of viral particles.

Lentiviruses are a valuable research tool for exploring gene function; however, researchers may wish to avoid production of pantropic lentivirus encoding known or suspected oncogenes. As an alternative, we present a safer protocol for use of ecotropic lentivirus on human cells modified to express the ecotropic receptor mSlc7a1.

Transdução de células humanas com Polymer-complexado Lentivirus Ecotropic para reforço de Biossegurança

Stem and tumor cell biology studies often require viral transduction of human cells with known or suspected oncogenes, raising major safety issues for ...

Induction and Analysis of Epithelial to Mesenchymal Transition

Epithelial to mesenchymal transition (EMT) is essential for proper morphogenesis during development. Misregulation of this process has been implicated as a key event in fibrosis and the progression of carcinomas to a metastatic state. Understanding the processes that underlie EMT is imperative for the early diagnosis and clinical control of these disease states. Reliable induction of EMT in vitro is a useful tool for drug discovery as well as to identify common gene expression signatures for diagnostic purposes. Here we demonstrate a straightforward method for the induction of EMT in a variety of cell types. Methods for the analysis of cells pre- and post-EMT induction by immunocytochemistry are also included. Additionally, we demonstrate the effectiveness of this method through antibody-based array analysis and migration/invasion assays.

A straightforward method is described for the induction of epithelial to mesenchymal transition (EMT) in a variety of cell types. Methods for the analysis of cells in EMT states by immunocytochemistry are included.

Indução e Análise de epitelial para mesenquimal Transição

Epithelial  to mesenchymal transition (EMT) is essential for proper morphogenesis during  development. Misregulation of this  process has been implicated ...

Primary Culture of Rat Adrenocortical Cells and Assays of Steroidogenic Functions

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary cultured rat adrenal cells and related functional assays (immunofluorescence staining of lipid droplet surface protein, as well as corticosterone analysis). Unlike an in vivo model, the variation of interexperiments in adrenal monolayer cultures is less and the experimental condition is easy to control. Besides, the source of rats is also more stable than other animals, like bovine ones. There are also several human adrenal cell lines (NCI-H295, NCI-H295R, SW13, etc.) that can be used in adrenal studies. However, the steroid production of these lines will still be influenced by numerous factors, which include serum lot number, passage number, mutant/loss of distinct genes, etc. Except for lacking 17&#945;-hydroxylase, the primary culture of rat adrenocortical cells is a better and more convenient technique for studying adrenal physiology. In summary, primary rat adrenal cultures could be a good in vitro platform for researchers to investigate the mechanisms of the reagent of interest in the adrenal gland system.

The hormone secreted by the adrenal cortex is vital for animals against stress and diseases. Here, we present a protocol to culture the primary rat adrenal cells. It could be a good in vitro platform for investigating the mechanisms of the reagent of interest in adrenal steroidogenesis and lipid biosynthesis.

Cultura primária de células Adrenocortical rato e ensaios das funções reguladora

The hormone which the adrenal cortex secretes is vital for animals against stress and diseases. The method described here is the procedure of primary ...

Multicellular Human Alveolar Model Composed of Epithelial Cells and Primary Immune Cells for Hazard Assessment

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II cells and two types of immune cells (i.e., human monocyte-derived macrophages [MDMs] and dendritic cells [MDDCs]). A protocol for assembling the multicellular model is provided. Alveolar epithelial cells (A549 cell line) are grown and differentiated under submerged conditions on permeable inserts in two-chamber wells, then combined with differentiated MDMs and MDDCs. Finally, the cells are exposed to an air-liquid interface for several days. As human primary immune cells need to be isolated from human buffy coats, immune cells differentiated from either fresh or thawed monocytes are compared in order to tailor the method based on experimental needs. The three-dimensional models, composed of alveolar cells with either freshly isolated or thawed monocyte-derived immune cells, show a statistically significant increase in cytokine (interleukins 6 and 8) release upon exposure to proinflammatory stimuli (lipopolysaccharide and tumor necrosis factor &#945;) compared to untreated cells. On the other hand, there is no statistically significant difference between the cytokine release observed in the cocultures. This shows that the presented model is responsive to proinflammatory stimuli in the presence of MDMs and MDDCs differentiated from fresh or thawed peripheral blood monocytes (PBMs). Thus, it is a powerful tool for investigations of acute biological response to different substances, including aerosolized drugs or nanomaterials.

Presented here is a protocol for primary human blood monocyte isolation as well as their differentiation into macrophages and dendritic cells and assembly with epithelial cells into a multicellular human lung model. Biological responses of cocultures composed of immune cells differentiated from either freshly isolated or thawed monocytes, upon exposure to proinflammatory stimuli, are compared.

Modelo Alveolar Humano Multicelular Composto por Células Epiteliais e Células Imunes Primárias para Avaliação de Riscos

A human alveolar cell coculture model is described here for simulation of the alveolar epithelial tissue barrier composed of alveolar epithelial type II ...

Isolation and Enrichment of Human Lung Epithelial Progenitor Cells for Organoid Culture

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, epithelial progenitor cells can self-renew and generate differentiating progeny that exhibit cellular functions similar to those of their in vivo counterparts. Herein we describe a step-by-step protocol to isolate region-specific progenitors from human lung and generate 3D organoid cultures as an experimental and validation tool. We define proximal and distal regions of the lung with the goal of isolating region-specific progenitor cells. We utilized a combination of enzymatic and mechanical dissociation to isolate total cells from the lung and trachea. Specific progenitor cells were then fractionated from the proximal or distal origin cells using fluorescence associated cell sorting (FACS) based on cell type-specific surface markers, such as NGFR&#160;for sorting basal cells and HTII-280 for sorting alveolar type II cells. Isolated basal or alveolar type II progenitors were used to generate 3D organoid cultures. Both distal and proximal progenitors formed organoids with a colony forming efficiency of 9-13% in distal region and 7-10% in proximal region when plated 5000 cell/well on day 30. Distal organoids maintained HTII-280+ alveolar type II cells in culture whereas proximal organoids differentiated into ciliated and secretory cells by day 30. These 3D organoid cultures can be used as an experimental tool for studying the cell biology of lung epithelium and epithelial mesenchymal interactions, as well as for the development and validation of therapeutic strategies targeting epithelial dysfunction in a disease.

This article provides a detailed methodology for tissue dissociation and cellular fractionation approaches allowing enrichment of viable epithelial cells from proximal and distal regions of the human lung. Herein these approaches are applied for the functional analysis of lung epithelial progenitor cells through the use of 3D organoids culture models.

Isolamento e enriquecimento de células progenitoras epiteliais pulmonares humanas para a cultura organoide

Epithelial organoid models serve as valuable tools to study the basic biology of an organ system and for disease modeling. When grown as organoids, ...