1. MEA Preparação <ol><li> MEAs para uso em experimentos de eletroporação pode ser fabricado usando a técnica de fotolitografia padrão, como descrito anteriormente 18 ou comprados diretamente de fornecedores de fabricantes MEA como Sistemas Multi Channel <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> e MED Alpha Scientific, Inc. <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)</a> . Os MEAs utilizados em nossos experimentos foram fabricados em casa na instalação de sala limpa na Universidade Estadual do Arizona, que é administrado pelo Centro de Pesquisas do Estado Sólido Eletrônica (CSSER). Os MEAs utilizados em nossos experimentos teve 32 índio eletrodos de óxido de estanho em um 8 por 4 matriz com tamanhos de eletrodos de diâmetro 50-200 mM dentro de um vidro 1 cm de diâmetro interior também. O vidro bem foi colada sobre a MEA usando polidimetilsiloxano (PDMS). </li><li> Esteriliza-se o MEA em autoclave antes da célula seeding. Outros métodos de esterilização, tais como a imersão em etanol a 70%, tratamento com UV e tratamento com água quente, tal como descrito noutro local também pode ser utilizado, desde que não comprometam a integridade do MEA. </li><li> Transfira a MEA autoclavado a uma capela de fluxo laminar e colocá-lo em um estéril prato de poliestireno padrão Petri. Antes de semear as células da MEA, esterilizar a câmara de fluxo laminar, ligando a luz UV durante 20 min. Se o MEA é sensível à luz UV cobrir a placa de Petri contendo o MEA com folha de alumínio estéril enquanto o capuz de fluxo está sob iluminação UV. </li></ol> 2. As células de sementeira no MEA <ol><li> Células de colheita a partir de uma linha de células aderentes em execução de células de mamíferos utilizando tripsina / EDTA e elevá-las a uma concentração de 100-200 células / uL de 1 ml de meio de células de galvanização. Em nossas experiências, temos sido bem sucedidos em cultura de células NIH 3T3 linha de fibroblastos, células HeLa e linha primários neurônios corticais e do hipocampo no MEA. </li><li&gt; Coloque uma gota 20-30 ul dos meios de comunicação com as células da MEA. Transferir a placa de Petri com o MEA para a incubadora. É importante assegurar que o tamanho de gota de célula é suficientemente larga para abranger todos os eléctrodos do MEA. </li><li> 2 horas depois de semear as células transferir a placa de Petri com o MEA da incubadora a câmara de fluxo laminar e adicionar suavemente 400-500 ul de pré meios aquecidos (37 ° C) para o MEA bem. Verificar ao microscópio para garantir que as células que ainda estão presos e uniformemente distribuído sobre a superfície MEA. Transferir a volta MEA para a incubadora. O período de espera de 2 horas antes da adição de media garante um tempo suficiente para que as células adiram à superfície MEA. Além dos meios de comunicação celulares também logo após a semeadura das células pode lavar as células. Se as células não aderem bem no MEA, a sua superfície pode ser tratada com factores de fixação de células aspolylysine tal, fibronectina, laminina e para melhorar a adesão celular. Em nossos experimentos com células HeLa que normalmente tratam a superfície MEA com fibronectin (10 ug / ml) durante 1-2 horas. </li><li> 8-12 horas após a sementeira das células, executa 1-2 lavagens com PBS para remover as células mortas. Meio celular pode substituir cada 24-48 horas, conforme necessário. Numa experiência típica, as células são usualmente preparados para a transfecção 24-48 horas após a sementeira. Para manter a consistência na eficácia de electroporação, recomenda-se esperar que a cultura é de pelo menos 80% confluentes antes da electroporação. </li></ol> 3. Site-specific A transfecção de siRNA <ol><li> Preparar uma solução de ácido nucleico. Para a transfecção siRNA, preparar uma solução de 1-2 uM de electroporação em gelo siRNA tampão de electroporação gelado para atingir um volume final de 200-400 ul. Neste artigo de vídeo que demonstram a transfecção de células HeLa com Alexa 488 scrambled sequência conjugado de siRNA. </li><li> Transferir o MEA com as células a partir do incubador a capa de fluxo laminar estéril. Remover os meios de comunicação celular a partir da MEA e efectuar uma lavagem com PBS. Em seguida, adicione 200-400 ul do ice frio de 1 uM solução eletroporação de siRNA para o bem. </li><li> Aplicar anódicas quadrados impulsos elétricos para os eletrodos direcionados (Consulte o passo 4 para determinar os parâmetros ótimos do pulso elétrico). Em nossos experimentos utilizamos uma Instrumentos pragmáticas gerador de onda 2414A para gerar pulsos elétricos e um dual-inline-package (DIP) 16 clipe pino para fazer contato com as pastilhas de títulos MEA. O gerador de forma de onda pode ser ligado ao clipe DIP através de uma placa de circuito impresso para proporcionar demultiplexor selectividade dos eléctrodos individuais. Para o eléctrodo de referência, coloque um cátodo de aço nos meios de comunicação celular. , Baixando o eléctrodo de referência de aço nos meios de comunicação, é extremamente importante para assegurar que não entra em contacto com a superfície do MEA. Em contacto, pode provocar danos nas células e o MEA. Recomenda-se a diminuir o eléctrodo de referência para uma posição, de 1 mm acima das células. Um espaçador com uma altura de 1 mm pode ser colocada no poço para auxiliar no posicionamento da refeletrodo cia. Uma abordagem alternativa ao uso de um eletrodo de referência externo é usar eletrodos vizinhos como cátodo ânodo-pares. </li><li> Imediatamente após a aplicação dos impulsos eléctricos para os eléctrodos de alvo no MEA, transferir o MEA volta para a incubadora. Após um período de incubação de 5 min, colocar suavemente a 75% do tampão de electroporação, com pré-aquecido (37 ° C) meios de comunicação celular. Mais tarde, imagiologia fluorescente pode ser feito para confirmar a entrega de siRNA fluorescentemente marcado para as células. </li></ol> 4. Otimização de parâmetros eletroporação <ol><li> Para determinar os parâmetros óptimos de impulsos eléctricos para electroporação, as experiências de electroporação de design para um intervalo de valores de amplitude de pulso, duração do impulso, e o número de pulsos. Na nossa experiência com as células HeLa, observou-se que um impulso de tensão de amplitude única de 3 V a 9 V, e duração de 1 mseg e 10 mseg para um tamanho de 100 um eléctrodo levaram a electroporação com sucesso mimorte celular nimal. É nossa recomendação que um parâmetro ser variada de cada vez e os outros são mantidas constantes. Para quantificar a eficiência da electroporação para cada um dos parâmetros de pulso, usamos iodeto de propídio (PI), um corante impermeante celular que o material de manchas nucleico e metodologia de ensaio baseado em directo, tal como descrito nos passos de 4,2-4,5. </li><li> Estabelecer uma cultura de células sobre a MEA seguindo as instruções descritas nos passos 1 e 2. </li><li> Antes da electroporação preparar um pi 300-500, de 30 ug / ml de solução em tampão de PI electroporação frio e 400 ul de 4 uM calcien AM solução em PBS. </li><li> Transferir o MEA da incubadora para a câmara de fluxo laminar. Remover os meios de comunicação celular a partir da MEA e efectuar uma lavagem com PBS. Em seguida, adicione 300-500 ul do tampão de electroporação arrefecido em gelo com PI. </li><li> Aplicar pulsos elétricos com amplitude e duração do pulso desejado para os eletrodos direcionados (O equipamento utilizado em nossos experimentos para gerar e aplicar o elecpulso tric a MEA é descrito na etapa 3.3.) Para o eléctrodo de referência ou um eléctrodo de aço diminuir no tampão de electroporação no MEA bem ou utilizar o eléctrodo vizinho como referência. Ao aplicar selectivamente pulsos de amplitudes diferentes ou durações de eléctrodos diferentes experiências múltiplas pode ser realizada no mesmo dispositivo. Por exemplo, em um 32 eléctrodo de MEA, 8 experiências podem ser realizadas através da divisão dos 32 eléctrodos em 8 grupos, cada um consistindo de quatro eléctrodos. Cada um dos 8 grupos podem então ser estimuladas por um pulso de voltagem rectangular único de duração de 5 mseg com amplitudes variando em 3 V-6 série V em incrementos de 0,5 V e um grupo pode ser usado como um controlo. </li><li> Imediatamente após a aplicação dos impulsos de tensão transferir a MEA na incubadora durante 5 min. Depois, remover suavemente a 75% do tampão de electroporação da MEA bem e substituí-la por meio de células quentes. Transferir a volta MEA para a incubadora. </li><li> Após um período de incubação de 2-4 hrsubstituir o meio de células no MEA bem com 300-500 ul de calceína 4 mM AM solução em PBS. Transferir a volta MEA a incubadora durante mais 30 minutos. </li><li> A seguir à incubação das células com calceína AM, substituir a calceína AM solução no MEA bem com PBS, depois duas lavagens com PBS. Obtenção de imagens fluorescentes das células utilizando um microscópio óptico com com filtros para PI (excitação / emissão - 535 nm/617 nm) e calceína AM (excitação / emissão - 490 nm/520 nm). A captação de PI, devido a eletroporação, faz com que células de fluorescência vermelha ea AM Calcein faz as células que estão vivos para fluorescência verde. Três cenários possíveis que podem ser observados são: 1) celulares eletroporados e vivo fica fluorescente (vermelho e verde), 2) células vivas, mas não eletroporadas (será apenas verde fluorescente) e 3) células mortas devido a eletroporação (só fica fluorescente vermelha) . A viabilidade celular foi calculada como uma percentagem do número total de células vivas no eléctrodo e a electroporação efficiency foi calculada como uma percentagem do número total de células carregadas com PI / siRNA. </li></ol> 5. Resultados representativos A Figura 1 mostra a carga específica do local de células HeLa com PI. Pode-se observar que apenas as células do eléctrodo demonstram uma absorção de PI (Figura 1B). Um ensaio ao vivo realizada pós electroporação foi utilizado para avaliar a viabilidade das células (Figura 1A). A eficiência de electroporação e a viabilidade das células para o exemplo mostrado na Figura 1 são de 81,8% e 96,1%, respectivamente. A viabilidade celular elevada e a eficiência de electroporação pode ser conseguida através da optimização dos parâmetros de electroporação de pulso. Site-specific a transfecção de células Hela com siRNA fluorescentemente marcado para um pulso de electroporação optimizado (8 V, 1 ms) é mostrado na Figura 2. A eficiência de electroporação para o siRNA de 8 V, 1 ms foi encontrado para ser 74,4 ± 16,0% eA viabilidade celular foi encontrado para ser 87,9 ± 8,4% (todos os dados representados como a média ± desvio padrão, n = 5). <img alt="Figura 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> Figura 1. Entrega específica do local de PI em células HeLa num eléctrodo 200 uM (6 V, 5 ms). A) Calcien ensaio baseado vivo realizados dois electroporação pós hr. Células vivas são indicados por fluorescência verde. B) Vermelho de fluorescência demonstra a captação de PI por células sobre o eléctrodo. C) Uma imagem sobreposta de A e B. As setas brancas indicam células que são electroporated e viva. As setas pretas indicam que as células são mortas. O círculo branco em A, B e C indicam o contorno do eléctrodo. <img alt="Figura 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> Figura 2. Transfecção de células HeLa com scrambl fluorescentemente marcadoseqüência e de siRNA. A) Imagem de células HeLa sobre um eléctrodo de 100 uM transfectadas com siRNA Alexa 488 marcado (8 V, 1 ms). B) Imagem de núcleos de células coradas com DAPI. O círculo branco marca a extremidade do eléctrodo.

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Neste artigo de vídeo que demonstram a utilização de MEA por site-specific transfecção de células HeLa com scrambled sequência de siRNA. Uma das vantagens desta tecnologia é a sua aplicabilidade a diferentes linhas de células, incluindo linhas de células primárias. O nosso laboratório tem demonstrado previamente a utilização desta tecnologia para o site-specific transfecção de cultura primária do hipocampo neuronal de rato E18 dia de idade e células NIH-3T3 com sequências de siRNA mexidos e GFP plasm?...

<table border="1"><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários (opcional) </td></tr><tr><td> Meios celulares: Avançado MEM L-Glutamina 200 mM Penicilina / estreptomicina De soro fetal bovino </td><td> Gibco / Invitrogen HIMEDIA Laboratories / VWR Lonza Group Ltd. Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Composição celular de mídia: 2% de FBS Pennstrep, 2% de L-glutamina e 2% em Advance MEM </td></tr><tr><td> Tripsina EDTA </td><td> Mediatech, Inc. </td><td> 25-053-CL </td><td></td></tr><tr><td> PBS </td><td> Mediatech, Inc. </td><td> 21-040-CV </td><td></td></tr><tr><td> Alexa 488 e rodamina marcado mexidos sequsiRNA referência </td><td> Qiagen, Inc. </td><td> 1027292 </td><td></td></tr><tr><td> Tampão de electroporação </td><td> Biorad Laboratories </td><td> 165-2677 </td><td></td></tr><tr><td> Forma de onda do gerador </td><td> Pragmático </td><td> 2414A </td><td> Qualquer forma de onda / gerador de impulsos que pode fornecer os impulsos desejados podem ser usados. </td></tr></table>

high efficiency, site-specific transfection of adherent cells with sirna using microelectrode arrays (mea)

A descoberta da via de RNAi em eucariotos e subsequente desenvolvimento de agentes de RNAi, como siRNA e shRNA, ter alcançado um método poderoso para silenciar genes específicos 1-8 para genômica funcional e terapêutica. Um dos maiores desafios envolvidos em estudos de RNAi base é a entrega de agentes de RNAi para as células-alvo. As técnicas tradicionais de entrega não virais, tais como a electroporação em massa e métodos de transfecção químicos muitas vezes não têm o necessário controlo espacial sobre a entrega e proporcionar eficiências de transfecção pobres 9-12. Avanços recentes em métodos químicos, tais como a transfecção lipidos catiónicos, polímeros catiónicos e nanopartículas têm resultado em eficiências de transfecção altamente melhorados 13. No entanto, estas técnicas ainda não oferecem o controle espacial preciso sobre entrega que pode beneficiar imensamente miniaturizado tecnologias de alta capacidade, estudos de células individuais e investigação de interações célula-célula. nt &quot;&gt; Recentes avanços tecnológicos na entrega de genes permitiram alto rendimento a transfecção de células aderentes 14-23, a maioria dos quais utilizam electroporação microescala. electroporação microescala oferece controlo espaço-temporal preciso sobre o parto (até as células individuais) e tem sido demonstrado para obter eficiências elevadas 19, 24-26. Além disso, as abordagens baseadas em electroporação não necessitam de um longo período de incubação (normalmente de 4 horas) com siRNA e complexos de ADN, conforme necessário, em métodos químicos à base de transfecção e levar à entrada directa de ADN nu, e siRNA moléculas no citoplasma celular. Como consequência a expressão de genes pode ser alcançada tão cedo quanto seis horas após a transfecção 27. nosso laboratório tem demonstrado o uso de matrizes de microeletrodos (MEA) de sítio-específica de transfecção em culturas aderentes de células de mamíferos 17-19. Na abordagem baseada MEA, a entrega de carga genética é conseguido através de micro-escala localizada electroporatina de células. Uma aplicação de impulsos eléctricos para os eléctrodos seleccionados gera campo eléctrico local que conduz a electroporação de células presentes na região dos eléctrodos estimulados. O controlo independente dos micro-eléctrodos proporciona um controlo espacial e temporal sobre a transfecção e também permite que múltiplas experiências de transfecção à base a ser executada com a mesma cultura aumentando o rendimento e reduzindo experimental de cultura para cultura de variabilidade. Aqui nós descrevemos a montagem experimental e do protocolo para transfecção alvo de células HeLa aderentes com uma seqüência de siRNA fluorescente etiquetado mexidos com eletroporação. O mesmo protocolo pode também ser utilizado para a transfecção de vectores plasmídicos. Além disso, o protocolo aqui descrito pode ser facilmente alargada a uma variedade de linhas de células de mamíferos, com pequenas modificações. Disponibilidade comercial de MEAs com padrões de eletrodos tanto pré-definidos e personalizados fazer esta t técnica acessívela maioria dos laboratórios de pesquisa Ø com equipamento básico de cultura de células.

Watch this Scientific Journal Video about High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA at JoVE.com

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Os detalhes do protocolo de artigo para site-specific transfecção de scrambled sequência de siRNA em uma cultura de células aderentes mamíferos usando uma rede de microeletrodos (MEA).

Alta eficiência, Transfecção site-specific de células aderentes com siRNA Usando matrizes de microeletrodos (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

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Research

JoVE Journal

Bioengineering

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (MEA)

13.4K visualizações.  Arizona State University. Os detalhes do protocolo de artigo para site-specific transfecção de scrambled sequência de siRNA em uma cultura de células aderentes mamíferos usando uma rede de microeletrodos (MEA).

Vídeo: High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays MEA

Therapeutic Gene Delivery and Transfection in Human Pancreatic Cancer Cells using Epidermal Growth Factor Receptor-targeted Gelatin Nanoparticles

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. Urgent need exists to develop novel clinically-translatable therapeutic strategies that can improve on the dismal survival statistics of pancreatic cancer patients. Although gene therapy in cancer has shown a tremendous promise, the major challenge is in the development of safe and effective delivery system, which can lead to sustained transgene expression. 
Gelatin is one of the most versatile natural biopolymer, widely used in food and pharmaceutical products. Previous studies from our laboratory have shown that type B gelatin could physical encapsulate DNA, which preserved the supercoiled structure of the plasmid and improved transfection efficiency upon intracellular delivery. By thiolation of gelatin, the sulfhydryl groups could be introduced into the polymer and would form disulfide bond within nanoparticles, which stabilizes the whole complex and once disulfide bond is broken due to the presence of glutathione in cytosol, payload would be released 2-5. Poly(ethylene glycol) (PEG)-modified GENS, when administered into the systemic circulation, provides long-circulation times and preferentially targets to the tumor mass due to the hyper-permeability of the neovasculature by the enhanced permeability and retention effect 6. Studies have shown over-expression of the epidermal growth factor receptor (EGFR) on Panc-1 human pancreatic adenocarcinoma cells 7. In order to actively target pancreatic cancer cell line, EGFR specific peptide was conjugated on the particle surface through a PEG spacer.8
Most anti-tumor gene therapies are focused on administration of the tumor suppressor genes, such as wild-type p53 (wt-p53), to restore the pro-apoptotic function in the cells 9. The p53 mechanism functions as a critical signaling pathway in cell growth, which regulates apoptosis, cell cycle arrest, metabolism and other processes 10. In pancreatic cancer, most cells have mutations in p53 protein, causing the loss of apoptotic activity. With the introduction of wt-p53, the apoptosis could be repaired and further triggers cell death in cancer cells 11. 
Based on the above rationale, we have designed EGFR targeting peptide-modified thiolated gelatin nanoparticles for wt-p53 gene delivery and evaluated delivery efficiency and transfection in Panc-1 cells.

Type B gelatin-based engineered nanovectors system (GENS) was developed for systemic gene delivery and transfection in the treatment of pancreatic cancer. By modification with epidermal growth factor receptor (EGFR) specific peptide on the surface of nanparticles, they could target on EGFR receptor and release plasmid under reducing environment, such as high intracellular glutathione concentrations.

Entrega de genes e Transfection em células pancreáticas humanas Cancer usando Fator de Crescimento Epidérmico Receptor-alvo à base de gelatina Nanovectors Engineered

More than 32,000 patients are diagnosed with pancreatic cancer in the United States per year and the disease is associated with very high mortality 1. ...

Bioengenharia

Site-specific Bacterial Chromosome Engineering: &#934;C31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as plasmid replication, plasmid stability, plasmid incompatibility, and plasmid copy number variance. This method uses the site-specific integrase from the Streptomyces phage (&Phi;) C312,3. The &Phi;C31 integrase catalyzes a direct recombination between two specific DNA sites: attB and attP (34 and 39 bp, respectively)4. This recombination is stable and does not revert5. A "landing pad" (LP) sequence consisting of a spectinomycin- resistance gene, aadA (SpR), and the E. coli &szlig;-glucuronidase gene (uidA) flanked by attP sites has been integrated into the chromosomes of Sinorhizobium meliloti, Ochrobactrum anthropi, and Agrobacterium tumefaciens in an intergenic region, the ampC locus, and the tetA locus, respectively. S. meliloti is used in this protocol. Mobilizable donor vectors containing attB sites flanking a stuffer red fluorescent protein (rfp) gene and an antibiotic resistance gene have also been constructed. In this example the gentamicin resistant plasmid pJH110 is used. The rfp gene6 may be replaced with a desired construct using SphI and PstI. Alternatively a synthetic construct flanked by attB sites may be sub-cloned into a mobilizable vector such as pK19mob7. The expression of the &Phi;C31 integrase gene (cloned from pHS628) is driven by the lac promoter, on a mobilizable broad host range plasmid pRK78139.
A tetraparental mating protocol is used to transfer the donor cassette into the LP strain thereby replacing the markers in the LP sequence with the donor cassette. These cells are trans-integrants. Trans-integrants are formed with a typical efficiency of 0.5%. Trans-integrants are typically found within the first 500-1,000 colonies screened by antibiotic sensitivity or blue-white screening using 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid (X-gluc). This protocol contains the mating and selection procedures for creating and isolating trans-integrants.

Site-specific Bacterial Chromosome Engineering: &amp;#934;C31 Integrase Mediated Cassette Exchange (IMCE)

A quick and efficient method to integrate foreign DNA of interest into pre-made acceptor strains, termed landing pad strains, is described. The method allows site-specific integration of a DNA cassette into the engineered landing pad locus of a given strain, through conjugation and expression of the &#934;C31 integrase.

Site-specific Engenharia cromossomo bacteriano: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

Engineering Adherent Bacteria by Creating a Single Synthetic Curli Operon

The method described here consists in redesigning E. coli adherence properties by assembling the minimum number of curli genes under the control of a strong and metal-overinducible promoter, and in visualizing and quantifying the resulting gain of bacterial adherence. This method applies appropriate engineering principles of abstraction and standardization of synthetic biology, and results in the BBa_K540000 Biobrick (Best new Biobrick device, engineered, iGEM 2011).
The first step consists in the design of the synthetic operon devoted to curli overproduction in response to metal, and therefore in increasing the adherence abilities of the wild type strain. The original curli operon was modified in silico in order to optimize transcriptional and translational signals and escape the "natural" regulation of curli. This approach allowed to test with success our current understanding of curli production. Moreover, simplifying the curli regulation by switching the endogenous complex promoter (more than 10 transcriptional regulators identified) to a simple metal-regulated promoter makes adherence much easier to control.
The second step includes qualitative and quantitative assessment of adherence abilities by implementation of simple methods. These methods are applicable to a large range of adherent bacteria regardless of biological structures involved in biofilm formation. Adherence test in 24-well polystyrene plates provides a quick preliminary visualization of the bacterial biofilm after crystal violet staining. This qualitative test can be sharpened by the quantification of the percentage of adherence. Such a method is very simple but more accurate than only crystal violet staining as described previously 1 with both a good repeatability and reproducibility. Visualization of GFP-tagged bacteria on glass slides by fluorescence or laser confocal microscopy allows to strengthen the results obtained with the 24-well plate test by direct observation of the phenomenon.

The design of a synthetic operon encoding both the secretory apparatus and the structural monomers of curli fibers is described. Overproduction of these amyloids and adherent polymers allows a measurable gain of adherence of the E. coli chassis1. Easy ways to visualize and quantify adherence are explained.

Engenharia bactérias aderentes pela criação de um único sintético Curli Operon

The  method described here consists in redesigning E. coli adherence properties by assembling the minimum number of  curli genes under the control of a ...

Ordering Single Cells and Single Embryos in 3D Confinement: A New Device for High Content Screening

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening based on cells in such environments have therefore serious limitations: cell organelles show extreme phenotypes, cell morphologies and sizes are heterogeneous and/or specific cell organelles cannot be properly visualized. In addition, cells in vivo are located in a 3D environment; in this situation, cells show different phenotypes mainly because of their interaction with the surrounding extracellular matrix of the tissue. In order to standardize and generate order of single cells in a physiologically-relevant 3D environment for cell-based assays, we report here the microfabrication and applications of a device for in vitro 3D cell culture. This device consists of a 2D array of microcavities (typically 105 cavities/cm2), each filled with single cells or embryos. Cell position, shape, polarity and internal cell organization become then normalized showing a 3D architecture. We used replica molding to pattern an array of microcavities, &#8216;eggcups&#8217;, onto a thin polydimethylsiloxane (PDMS) layer adhered on a coverslip. Cavities were covered with fibronectin to facilitate adhesion. Cells were inserted by centrifugation. Filling percentage was optimized for each system allowing up to 80%. Cells and embryos viability was confirmed. We applied this methodology for the visualization of cellular organelles, such as nucleus and Golgi apparatus, and to study active processes, such as the closure of the cytokinetic ring during cell mitosis. This device allowed the identification of new features, such as periodic accumulations and inhomogeneities of myosin and actin during the cytokinetic ring closure and compacted phenotypes for Golgi and nucleus alignment. We characterized the method for mammalian cells, fission yeast, budding yeast, C. elegans with specific adaptation in each case. Finally, the characteristics of this device make it particularly interesting for drug screening assays and personalized medicine.

We report a device and a new method to study cells and embryos. Single cells are precisely ordered in microcavity arrays. Their 3D confinement is a step towards 3D environments encountered in physiological conditions and allows organelle orientation. By controlling cell shape, this setup minimizes variability reported in standard assays.

Ordenando células individuais e único embriões em 3D Confinamento: Um novo dispositivo de alta Screening conteúdo

Biological cells are usually observed on flat (2D) surfaces. This condition is not physiological, and phenotypes and shapes are highly variable. Screening ...

Porous Silicon Microparticles for Delivery of siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the successful implementation of siRNA therapy requires the use of delivery platforms that can overcome the major challenges of siRNA delivery, such as enzymatic degradation, low intracellular uptake and lysosomal entrapment. Here, a protocol for the preparation and use of a biocompatible and effective siRNA delivery system is presented. This platform consists of polyethylenimine (PEI) and arginine (Arg)-grafted porous silicon microparticles, which can be loaded with siRNA by performing a simple mixing step. The silicon particles are gradually degraded over time, thereby triggering the formation of Arg-PEI/siRNA nanoparticles. This delivery vehicle provides a means for protecting and internalizing siRNA, without causing cytotoxicity. The major steps of polycation functionalization, particle characterization, and siRNA loading are outlined in detail. In addition, the procedures for determining particle uptake, cytotoxicity, and transfection efficacy are also described.

Delivery remains the main challenge for the therapeutic implementation of small interfering RNA (siRNA). This protocol involves the use of a multifunctional and biocompatible siRNA delivery platform, consisting of arginine and polyethylenimine grafted porous silicon microparticles.

Porous Silicon micropartículas para entrega de siRNA Therapeutics

Small interfering RNA (siRNA) can be used to suppress gene expression, thereby providing a new avenue for the treatment of various diseases. However, the ...

Delivery of Therapeutic siRNA to the CNS Using Cationic and Anionic Liposomes

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes. The emergence of prion diseases in wildlife populations and their increasing threat to human health has led to increased efforts to find a treatment for these diseases. Recent studies have found numerous anti-prion compounds that can either inhibit the infectious PrPRes isomer or down regulate the normal cellular prion protein. However, most of these compounds do not cross the blood brain barrier to effectively inhibit PrPRes formation in brain tissue, do not specifically target neuronal PrPC, and are often too toxic to use in animal or human subjects. 
We investigated whether siRNA delivered intravascularly and targeted towards neuronal PrPC is a safer and more effective anti-prion compound. This report outlines a protocol to produce two siRNA liposomal delivery vehicles, and to package and deliver PrP siRNA to neuronal cells. The two liposomal delivery vehicles are 1) complexed-siRNA liposome formulation using cationic liposomes (LSPCs), and 2) encapsulated-siRNA liposome formulation using cationic or anionic liposomes (PALETS). For the LSPCs, negatively charged siRNA is electrostatically bound to the cationic liposome. A positively charged peptide (RVG-9r [rabies virus glycoprotein]) is added to the complex, which specifically targets the liposome-siRNA-peptide complexes (LSPCs) across the blood brain barrier (BBB) to acetylcholine expressing neurons in the central nervous system (CNS). For the PALETS (peptide addressed liposome encapsulated therapeutic siRNA), the cationic and anionic lipids were rehydrated by the PrP siRNA. This procedure results in encapsulation of the siRNA within the cationic or anionic liposomes. Again, the RVG-9r neuropeptide was bound to the liposomes to target the siRNA/liposome complexes to the CNS. Using these formulations, we have successfully delivered PrP siRNA to AchR-expressing neurons, and decreased the PrPC expression of neurons in the CNS.

The goal of this protocol is to use cationic/anionic liposomes with a neuro-targeting peptide as a CNS delivery system to enable siRNA to cross the BBB. The optimization of a delivery system for treatments, like siRNA, would allow for more treatment options for prion and other neurodegenerative diseases.

Entrega de terapêutica siRNA para o SNC Usando catiônicos e aniônicos lipossomas

Prion diseases result from the misfolding of the normal, cellular prion protein (PrPC) to an abnormal protease resistant isomer called PrPRes.  The ...

Comprehensive Evaluation of the Effectiveness and Safety of Placenta-Targeted Drug Delivery Using Three Complementary Methods

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to the placenta while minimizing fetal and maternal side effects remains challenging. Targeted nanoparticle carriers provide new opportunities to treat placental disorders. We recently demonstrated that a synthetic placental chondroitin sulfate A binding peptide (plCSA-BP) could be used to guide nanoparticles to deliver drugs to the placenta. In this protocol, we describe in detail a system for assessing the efficiency of drug delivery to the placenta by plCSA-BP that employs three separate methods used in combination: in vivo imaging, high-frequency ultrasound (HFUS), and high-performance liquid chromatography (HPLC). Using in vivo imaging, plCSA-BP-guided nanoparticles were visualized in the placentas of live animals, while HFUS and HPLC demonstrated that plCSA-BP-conjugated nanoparticles efficiently and specifically delivered methotrexate to the placenta. Thus, a combination of these methods can be used as an effective tool for the targeted delivery of drugs to the placenta and development of new treatment strategies for several pregnancy complications.

We describe a system that utilizes three methods to evaluate the safety and effectiveness of placenta-targeted drug delivery: in vivo imaging to monitor nanoparticle accumulation, high-frequency ultrasound to monitor placental and fetal development, and HPLC to quantify drug delivery to tissue.

Avaliação global da eficácia e segurança de entrega de droga Placenta-alvo usando três métodos complementares

No effective treatments currently exist for placenta-associated pregnancy complications, and developing strategies for the targeted delivery of drugs to ...

Interfacing Microfluidics with Microelectrode Arrays for Studying Neuronal Communication and Axonal Signal Propagation

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of electrophysiological activity for long periods. However, the property of sensing signals from all sources around every microelectrode can become unfavorable when trying to understand communication and signal propagation in neuronal circuits. In a neuronal network, several neurons can be simultaneously activated and can generate overlapping action potentials, making it difficult to discriminate and track signal propagation. Considering this limitation, we have established an in vitro setup focused on assessing electrophysiological communication, which is able to isolate and amplify axonal signals with high spatial and temporal resolution. By interfacing microfluidic devices and MEAs, we are able to compartmentalize neuronal cultures with a well-controlled alignment of the axons and microelectrodes. This setup allows recordings of spike propagation with a high signal-to-noise ratio over the course of several weeks. Combined with specialized data analysis algorithms, it provides detailed quantification of several communication related properties such as propagation velocity, conduction failure, firing rate, anterograde spikes, and coding mechanisms.
This protocol demonstrates how to create a compartmentalized neuronal culture setup over substrate-integrated MEAs, how to culture neurons in this setup, and how to successfully record, analyze and interpret the results from such experiments. Here, we show how the established setup simplifies the understanding of neuronal communication and axonal signal propagation. These platforms pave the way for new in vitro models with engineered and controllable neuronal network topographies. They can be used in the context of homogeneous neuronal cultures, or with co-culture configurations where, for example, communication between sensory neurons and other cell types is monitored and assessed. This setup provides very interesting conditions to study, for example, neurodevelopment, neuronal circuits, information coding, neurodegeneration and neuroregeneration approaches.

This protocol aims to demonstrate how to combine in vitro microelectrode arrays with microfluidic devices for studying action potential transmission in neuronal cultures. Data analysis, namely the detection and characterization of propagating action potentials, is performed using a new advanced, yet user-friendly and freely available, computational tool.

Microfluídica interfaceamento com matrizes de microeletrodos para estudar comunicação Neuronal e propagação de sinal Axonal

Microelectrode arrays (MEAs) are widely used to study neuronal function in vitro. These devices allow concurrent non-invasive recording/stimulation of ...

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural underpinnings of brain disease and injury. As electrodes are miniaturized to the scale of individual cells, a corresponding rise in the interface impedance limits the quality of recorded signals. Additionally, conventional electrode materials are stiff, resulting in a significant mechanical mismatch between the electrode and the surrounding brain tissue, which elicits an inflammatory response that eventually leads to a degradation of the device performance. To address these challenges, we have developed a process to fabricate flexible microelectrodes based on Ti3C2 MXene, a recently discovered nanomaterial that possesses remarkably high volumetric capacitance, electrical conductivity, surface functionality, and processability in aqueous dispersions. Flexible arrays of Ti3C2 MXene microelectrodes have remarkably low impedance due to the high conductivity and high specific surface area of the Ti3C2 MXene films, and they have proven to be exquisitely sensitive for recording neuronal activity. In this protocol, we describe a novel method for micropatterning Ti3C2 MXene into microelectrode arrays on flexible polymeric substrates and outline their use for in vivo micro-electrocorticography recording. This method can easily be extended to create MXene electrode arrays of arbitrary size or geometry for a range of other applications in bioelectronics and it can also be adapted for use with other conductive inks besides Ti3C2 MXene. This protocol enables simple and scalable fabrication of microelectrodes from solution-based conductive inks, and specifically allows harnessing the unique properties of hydrophilic Ti3C2 MXene to overcome many of the barriers that have long hindered the widespread adoption of carbon-based nanomaterials for high-fidelity neural microelectrodes.

Fabrication of Ti3C2 MXene Microelectrode Arrays for In Vivo Neural Recording

We describe here a method for fabricating Ti3C2 MXene microelectrode arrays and utilizing them for in vivo neural recording.

Fabricação de Matrizes de microeletrodo Ti3C2 MXene para gravação neural in vivo

Implantable microelectrode technologies have been widely used to elucidate neural dynamics at the microscale to gain a deeper understanding of the neural ...

Fabrication of 3D Cardiac Microtissue Arrays using Human iPSC-Derived Cardiomyocytes, Cardiac Fibroblasts, and Endothelial Cells

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided a unique opportunity to study the complex interplay among different cardiovascular cell types that drives tissue development and disease. In the area of cardiac tissue models, several sophisticated three-dimensional (3D) approaches use induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) to mimic physiological relevance and native tissue environment with a combination of extracellular matrices and crosslinkers. However, these systems are complex to fabricate without microfabrication expertise and require several weeks to self-assemble. Most importantly, many of these systems lack vascular cells and cardiac fibroblasts that make up over 60% of the nonmyocytes in the human heart. Here we describe the derivation of all three cardiac cell types from iPSCs to fabricate cardiac microtissues. This facile replica molding technique allows cardiac microtissue culture in standard multi-well cell culture plates for several weeks. The platform allows user-defined control over microtissue sizes based on initial seeding density and requires less than 3 days for self-assembly to achieve observable cardiac microtissue contractions. Furthermore, the cardiac microtissues can be easily digested while maintaining high cell viability for single-cell interrogation with the use of flow cytometry and single-cell RNA sequencing (scRNA-seq). We envision that this in vitro model of cardiac microtissues will help accelerate validation studies in drug discovery and disease modeling.

Here, we describe an easy-to-use methodology to generate 3D self-assembled cardiac microtissue arrays composed of pre-differentiated human-induced pluripotent stem cell-derived cardiomyocytes, cardiac fibroblasts, and endothelial cells. This user-friendly and low cell requiring technique to generate cardiac microtissues can be implemented for disease modeling and early stages of drug development.

Fabricação de matrizes microtissue cardíacas 3D usando Cardiomiócitos derivados do iPSC humano, fibroblastos cardíacos e células endoteliais

Generation of human cardiomyocytes (CMs), cardiac fibroblasts (CFs), and endothelial cells (ECs) from induced pluripotent stem cells (iPSCs) has provided ...