1. MEA Preparación <ol><li> MEA para su uso en experimentos de electroporación puede ser fabricado utilizando la técnica de fotolitografía estándar como se describe anteriormente 18 o comprados directamente a los vendedores de los fabricantes de MEA, tales como sistemas de canales múltiples <a href="http://www.multichannelsystems.com" target="_blank">(http:/www.multichannelsystems.com/)</a> y alfa MED Scientific Inc., <a href="http://www.med64.com/" target="_blank">( http://www.MED64.com)</a> . Los AMUMA utilizados en nuestros experimentos se fabrican en el local en la sala limpia en Arizona State University, el cual es administrado por el Centro de Investigación Electrónica de estado sólido (CSSER). Los AMUMA utilizados en nuestros experimentos con 32 electrodos de óxido de estaño e indio en un 8 por 4 matriz con diámetros de electrodos 50 a 200 micras dentro de un vaso 1 cm de diámetro interior también. El vidrio también se unió a la MEA polidimetilsiloxano utilizando (PDMS). </li><li> Esterilizar el MEA en autoclave antes de célula seeding. Otros métodos de esterilización tal como un remojo en 70% de etanol, tratamiento UV y tratamiento con agua caliente como se describe en otra parte también se puede utilizar siempre y cuando no comprometan la integridad de la MEA. </li><li> Transferir el autoclave MEA a una campana de flujo laminar y colocarlo en un patrón de poliestireno placa de Petri estéril. Antes de la siembra de células sobre la MEA, esterilizar la campana de flujo laminar mediante la activación de la luz UV durante 20 min. Si el MEA es sensible a la luz UV cubrir la placa de Petri que contiene el MEA con papel de aluminio estéril mientras que la campana de flujo es bajo iluminación UV. </li></ol> 2. Las células siembra sobre el MEA <ol><li> Cosecha células de una línea celular adherente de funcionamiento de las células de mamífero utilizando tripsina / EDTA y elevarlas a una concentración de 100-200 células / l en 1 ml de medio de células para la siembra. En nuestros experimentos hemos tenido éxito en el cultivo de línea celular NIH 3T3 fibroblastos, la línea de células HeLa y neuronas corticales primarias del hipocampo y en el MEA. </li><li&gt; Coloque una gota 20-30 l de medios de comunicación con las celdas de la MEA. Transferir la placa de Petri con el MEA a la incubadora. Es importante asegurarse de que el tamaño de la gota de células es lo suficientemente grande como para cubrir todos los electrodos en el MEA. </li><li> 2 horas después de la siembra de las células transferir la placa de Petri con el MEA de la incubadora para campana de flujo laminar y suavemente añadir 400-500 l de preparación de soportes calentado (37 ° C) a la MEA bien. Ver bajo el microscopio para asegurar que las células están todavía unido y distribuida uniformemente en la superficie de MEA. Transferir la parte posterior MEA a la incubadora. El período de 2 hr de espera antes de la adición de medios de comunicación asegura un tiempo suficiente para que las células se adhieran a la superficie de MEA. La adición de medios de células demasiado pronto después de la siembra de las células puede lavar las células. Si las células no se adhieren bien sobre la MEA, su superficie puede ser tratada con factores de unión celular, tales aspolylysine, fibronectina y laminina para mejorar la adhesión celular. En nuestros experimentos con células HeLa que solemos tratar la superficie con MEA fibronectin (10 ug / ml) durante 1-2 hr. </li><li> 8-12 horas después de la siembra de las células, realizar 1-2 lavados con PBS para eliminar las células muertas. Medio celular puede cambiarse cada 24-48 horas, según sea necesario. En un experimento típico, las células generalmente están listos para la transfección 24-48 h después de la siembra. Para mantener la coherencia en la eficiencia de la electroporación, se recomienda que esperar hasta que el cultivo es de al menos 80% confluentes antes de la electroporación. </li></ol> 3. Específica de sitio La transfección de siRNA <ol><li> Preparar la solución de ácido nucleico. Para la transfección siRNA, preparar un siRNA 1-2 mM solución electroporación en tampón de electroporación enfriado en hielo para alcanzar un volumen final de 200-400 l. En este artículo de vídeo se demuestra la transfección de células HeLa con Alexa 488 conjugada revueltos secuencia de siRNA. </li><li> Transferir la MEA con células de la incubadora a la campana de flujo laminar estéril. Eliminar los medios celulares a partir de la MEA y realizar un lavado con PBS. Después de eso, añadir 200-400 l de la ice frío 1 mM siRNA electroporación solución al pozo. </li><li> Aplicar anódicos impulsos eléctricos a los electrodos cuadrados específicas (Consulte el paso 4 para determinar los parámetros óptimos de los impulsos eléctricos). En nuestros experimentos hemos utilizado algunos instrumentos pragmáticos 2414a generador de forma de onda para generar impulsos eléctricos y una dual-inline-paquetes (DIP) 16 pin clip de entrar en contacto con los terminales de unión de los AMUMA. El generador de forma de onda puede ser conectado a la pinza DIP a través de una placa de circuito impreso demultiplexor para proporcionar selectividad de los electrodos individuales. Para el electrodo de referencia, colocar un cátodo de acero en los medios celulares. Aunque bajar el electrodo de referencia de acero en los medios de comunicación es extremadamente importante para asegurarse de que no hace contacto con la superficie del MEA. Tras el contacto, puede causar daño a las células y la MEA. Se recomienda para bajar el electrodo de referencia a una posición de 1 mm por encima de las células. Un separador con una altura de 1 mm puede ser colocado en el pozo para ayudar en la colocación de la refrencia electrodo. Un enfoque alternativo a la utilización de un electrodo de referencia externo es el uso de electrodos vecinos como pares de ánodo cátodo. </li><li> Inmediatamente después de aplicar los impulsos eléctricos a los electrodos de destino de la MEA, transferir la parte posterior MEA a la incubadora. Después de un período de incubación de 5 min, suavemente reemplazar 75% del tampón de electroporación con pre-calentado (37 ° C) medios celulares. Más tarde, fluorescente de imágenes puede realizarse para confirmar la entrega de ARNsi marcado fluorescentemente a las células. </li></ol> 4. Electroporación optimización de parámetros <ol><li> Para determinar los parámetros óptimos de impulsos eléctricos para electroporación, diseñar experimentos de electroporación para una gama de valores de amplitud de pulso, duración del pulso, y el número de pulsos. En nuestra experiencia con células HeLa, se observó que un pulso de voltaje único de la amplitud de 3 V a 9 V, y la duración de 1 ms a 10 ms para un electrodo de tamaño de 100 micras llevaron a electroporación con éxito millasla muerte celular nimal. Es nuestra recomendación de que un parámetro puede variar a la vez y los demás se mantienen constantes. Para cuantificar la eficiencia de la electroporación para cada uno de los parámetros del pulso, se utiliza yoduro de propidio (PI), un colorante impermeant celular que el material de manchas nucleico, y la metodología de ensayo basado en directo, como se describe en los pasos 4.2-4.5. </li><li> Establecer un cultivo de células en el MEA siguiendo las instrucciones descritas en los pasos 1 y 2. </li><li> Antes de la electroporación preparar un 300-500 l de 30 mg / ml de solución PI en tampón de electroporación enfriada con hielo y 400 l de 4 mM calcien AM solución en PBS. </li><li> Transferir la MEA de la incubadora a la campana de flujo laminar. Eliminar los medios celulares a partir de la MEA y realizar un lavado con PBS. Después de eso, añadir 300-500 l del tampón de electroporación hielo frío con PI. </li><li> Aplicar impulsos eléctricos con la amplitud del pulso deseado y la duración de los electrodos dirigidos (El equipo usado en nuestros experimentos para generar y aplicar la electric pulso a la MEA se describe en el paso 3,3). Para el electrodo de referencia, bien bajar un electrodo de acero en el tampón de electroporación en la MEA o bien utilizar el electrodo vecino como referencia. Aplicando selectivamente impulsos de distintas amplitudes o duraciones de diferentes electrodos múltiples experimentos se pueden realizar en el mismo dispositivo. Por ejemplo, en un electrodo de 32 MEA, 8 experimentos se pueden realizar mediante el fraccionamiento de los 32 electrodos en 8 grupos, cada uno compuesto de 4 electrodos. Cada uno de los 8 grupos entonces puede ser estimulado por un pulso rectangular de una sola tensión de una duración de 5 ms con amplitudes que varían en 3 V-6 rango de V en incrementos de 0,5 V y un grupo se puede utilizar como un control. </li><li> Inmediatamente después de aplicar los impulsos de tensión transferir el MEA a la incubadora durante 5 min. Posteriormente, retirar suavemente el 75% del tampón de electroporación de la MEA bien y reemplazarlo con medios celulares cálidos. Transferir la parte posterior MEA a la incubadora. </li><li> Después de un periodo de incubación de 2-4 hrsustituir los medios celulares en el MEA bien con 300-500 l de 4 mM de calceína AM solución en PBS. Transferir la parte posterior MEA a la incubadora durante otros 30 min. </li><li> Después de la incubación de las células con calceína AM, sustituir la solución de calceína AM en el MEA bien con PBS después de dos lavados con PBS. Obtener imágenes fluorescentes de las células utilizando un microscopio óptico con filtros para PI (excitación / emisión - 535 nm/617 nm) y calceína AM (excitación / emisión - 490 nm/520 nm). La captación de PI, debido a la electroporación, hace que las células fluorescencia roja y la calceína AM hace que las células que están vivas que emite fluorescencia verde. Tres escenarios posibles que se pueden observar son: 1) celulares electroporadas y vivo será fluorescente (rojo y verde), 2) células vivas pero no electroporación (sólo presenta fluorescencia verde) y 3) células muertas debido a la electroporación (sólo presenta fluorescencia roja) . La viabilidad celular se calcula como un porcentaje del número total de células vivas en el electrodo y la electroporación efficiency se calculó como porcentaje del número total de células cargadas con PI / siRNA. </li></ol> 5. Los resultados representativos La Figura 1 muestra la carga específica del sitio de células HeLa con PI. Se puede observar que sólo las células en el electrodo de demostrar una captación de PI (Figura 1B). Un ensayo realizado en vivo después de la electroporación se utilizó para evaluar la viabilidad de las células (Figura 1A). La eficiencia de la electroporación y la viabilidad celular para el ejemplo mostrado en la Figura 1 son 81,8% y 96,1% respectivamente. Alta viabilidad celular y la eficiencia de la electroporación se puede lograr mediante la optimización de los parámetros del pulso de electroporación. Sitio específico de la transfección de células HeLa con ARNsi marcado fluorescentemente y para un pulso de electroporación optimizado (8 V, 1 ms) se muestra en la Figura 2. La eficiencia de la electroporación para el siRNA en V 8 mseg, 1 se encontró que era 74,4 ± 16,0% yla viabilidad celular se encontró que era 87,9 ± 8,4% (todos los datos representados como media ± desviación estándar, n = 5). <img alt="Figura 1" src="/files/ftp_upload/4415/4415fig1.jpg" /> Figura 1. Específica de sitio de entrega de PI en células HeLa en un electrodo m 200 (6 V, 5 mseg). A) calcien ensayo basado en directo interpretada 2 h después de la electroporación. Células vivas se indican con fluorescencia verde. B) Red de fluorescencia demuestra la captación de PI por las células en el electrodo. C) Una imagen superpuesta de A y B. Las flechas blancas indican que las células se someten a electroporación y vivo. Las flechas negras indican que las células están muertas. El círculo blanco en A, B y C indica el contorno del electrodo. <img alt="Figura 2" src="/files/ftp_upload/4415/4415fig2.jpg" /> Figura 2. Transfección de células HeLa con scrambl marcado fluorescentementee secuencia de siRNA. A) Imagen de células HeLa en un electrodo 100 micras transfectadas con siRNA Alexa 488 marcado (8 V, 1 mseg). B) Imagen de núcleos de células teñidas con DAPI. El círculo blanco marca el borde del electrodo.

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No hay conflictos de interés declarado.

En este artículo de vídeo que demuestran el uso de MEA para sitio específico de la transfección de células HeLa con revueltos secuencia de siRNA. Una de las ventajas de esta tecnología es su aplicabilidad a diferentes líneas celulares, incluyendo líneas celulares primarias. Nuestro laboratorio ha demostrado previamente la utilización de esta tecnología específica de sitio primario de la transfección de cultivo neuronal de hipocampo de rata E18 días de edad y células NIH-3T3 con revueltos secuencias de siRN...

<table border="1"><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios (opcional) </td></tr><tr><td> Medios de comunicación celulares: Advanced MEM L-glutamina 200 mM Penicilina / Estreptomicina El suero fetal bovino </td><td> Gibco / Invitrogen Himedia Laboratorios / VWR Lonza Group Ltd. Gibco / Invitrogen </td><td> 12492-013 95057-448 09-757F 16000-044 </td><td> Celular medios composición: 2% de FBS, Pennstrep 2% de L-glutamina y 2% en 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 y rodamina etiquetados revueltos SEQUcia siRNA </td><td> Qiagen, Inc. </td><td> 1027292 </td><td></td></tr><tr><td> La electroporación búfer </td><td> Biorad Laboratories </td><td> 165-2677 </td><td></td></tr><tr><td> Generador de forma de onda </td><td> Pragmático </td><td> 2414a </td><td> Cualquier forma de onda / generador de impulsos que puede entregar los pulsos deseado puede ser utilizado. </td></tr></table>

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

El descubrimiento de la vía de RNAi en eucariotas y el posterior desarrollo de los agentes de ARNi, como siRNA y shRNA, han logrado un método potente para el silenciamiento de genes específicos de 1-8 para la genómica funcional y la terapéutica. Un reto importante involucrado en estudios basados ​​en RNAi es el suministro de agentes de ARNi a células diana. Tradicionales técnicas no víricas de envío, tales como electroporación grandes cantidades y métodos químicos de transfección a menudo carecen del control necesario espacial sobre la entrega y proporcionar eficiencias de transfección 9-12 pobres. Los recientes avances en los métodos de transfección químicas tales como lípidos catiónicos, polímeros catiónicos y nanopartículas han dado lugar a eficiencias de transfección muy mejoradas 13. Sin embargo, estas técnicas aún no ofrecen un control preciso espacial sobre la entrega que se pueden beneficiar inmensamente miniaturizado tecnologías de alto rendimiento, estudios de células individuales y la investigación de las interacciones célula-célula. nt &quot;&gt; avances tecnológicos recientes en la administración génica han permitido alto rendimiento de transfección de células adherentes 14-23, una mayoría de los cuales utilizan electroporación microescala. electroporación Microscale ofrece espacio-temporal preciso control sobre la entrega (hasta células individuales) y se ha demostrado para lograr altas eficiencias de 19, 24-26. Además, los enfoques basados ​​en la electroporación no requieren un prolongado período de incubación (típicamente de 4 horas) con complejos de DNA y siRNA como sea necesario en los métodos químicos de transfección basados ​​en y conducir a la introducción directa de ADN desnudo y siRNA moléculas en el citoplasma de la célula. Como consecuencia la expresión génica se puede lograr tan pronto como seis horas después de la transfección 27. Nuestro laboratorio ha demostrado previamente la utilización de matrices de microelectrodos (MEA) para sitio específico de transfección en cultivos celulares de mamíferos adherentes 17-19. En el enfoque basado en MEA, entrega de carga genética se logra a través de micro-escala localizada electroporatien las células. Una aplicación de pulsos eléctricos a los electrodos seleccionados genera el campo eléctrico local que conduce a la electroporación de células presentes en la región de los electrodos estimulados. El control independiente de los microelectrodos proporciona un control espacial y temporal sobre la transfección y también permite múltiples experimentos de transfección basados ​​en que se realizará en el mismo cultivo aumentando la producción experimental y la reducción de la cultura a la cultura variabilidad. Aquí se describe la configuración experimental y el protocolo para la transfección selectiva de adherentes células HeLa con un siRNA secuencia de marcado con fluorescencia codificado utilizando electroporación. El mismo protocolo también puede ser utilizado para la transfección de vectores de plásmido. Además, el protocolo aquí descrito puede extenderse fácilmente a una variedad de líneas celulares de mamíferos, con modificaciones menores. Disponibilidad comercial de los acuerdos ambientales multilaterales con modelos de electrodos tanto predefinidos y personalizados que este t técnica accesibleo La mayoría de los laboratorios de investigación con equipo básico de cultivo celular.

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

El artículo detalla el protocolo específico de sitio de la transfección de revueltos secuencia de siRNA en un cultivo de células adherentes de mamíferos usando una matriz de microelectrodos (MEA).

Alta eficiencia, específica del sitio La transfección de células adherentes con siRNA uso de arrays de microelectrodos (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 ...

high-efficiency-site-specific-transfection-adherent-cells-with-sirna

Research

JoVE Journal

Bioengineering

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

13.4K vistas.  Arizona State University. El artículo detalla el protocolo específico de sitio de la transfección de revueltos secuencia de siRNA en un cultivo de células adherentes de mamíferos usando una matriz de microelectrodos (MEA).

Video: 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 y transfección de genes en células humanas de cáncer de páncreas con factor de crecimiento epidérmico receptor dirigidos a base de gelatina Nanovectors Ingeniería

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. ...

Investigación

Bioingeniería

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.

Sitio específico de Ingeniería cromosoma bacteriano: ΦC31 integrasa mediada 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.

Ingeniería bacterias adherentes mediante la creación de un único operón sintético curli

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 las células individuales y solo los embriones en 3D Confinamiento: un nuevo dispositivo para la selección de alto contenido

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.

Silicio Poroso micropartículas para la entrega de siRNA Terapéutica

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 Therapeutic siRNA al SNC Usando catiónicos y aniónicos Los liposomas

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.

Evaluación integral de la efectividad y seguridad de la entrega de drogas orientada a Placenta utilizando tres métodos complementarios

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 de entretela con arreglos de microelectrodos para el estudio de la comunicación Neuronal y Axonal señal propagación

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.

Fabricación de ti3C2 Matrizs de Microelectrodos MXene para Grabación Neuronal 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.

Fabricación de matrices de microtejidos cardíacos 3D utilizando cardiomiocitos humanos derivados de iPSC, fibroblastos cardíacos y células endoteliales

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