Para Margaret Smith CM por la amabilidad de proporcionar el clon de la integrasa Apoyo financiero de: Genome Canada / Genome Prairie NSERC Discovery y subvenciones de proyectos estratégicos

1. Producción de la Cultura <ol><li> Preparar los medios líquidos estériles: TY 10 (5 g / l de triptona, 3 g / l de extracto de levadura, 0,44 g / l de cloruro de calcio dihidrato), y LB 11 (10 g / l de triptona, 5 g / l de extracto de levadura, 5 g / cloruro de sodio, pH 7). </li><li> Inocular a partir de una sola colonia: SmUW227 (S. meliloti LP-deformación: la construcción se describe en otro lugar, la cepa detalles de la construcción están disponibles bajo petición) () en 5 ml de TY medios de comunicación con 50 mg / ml de espectinomicina. Inocular las siguientes cepas en 5 ml de medio líquido LB, que complementa con el antibiótico que se administra: E. E. DH5a 12 contiene pJC2, la expresión integrasa plásmido, con 10 mg / ml de tetraciclina, E. E. MT616 13, el activista, con 10 mg / ml de cloramfenicol, y E. E. DH5a contiene pJH110, el casete de plásmido donante, con 5 ug / ml de gentamicina. </li><li> Incubar E. E. noche a la mañana a 37 y las cepas°; C con agitación constante. Incubar S. meliloti cepa a 30 ° C durante dos días con agitación. Es posible obtener una S. meliloti noche a la mañana con un inóculo grande (1:500 subcultura de una cultura saturada) la cultura. </li></ol> 2. Cultura preparación y mezcla <ol><li> Lavar 1,5 ml de cultivo de cada cepa por recoger el sedimento celular por centrifugación a 17.000 xg durante 30 segundos y resuspender en 1 ml estéril 0,85% de NaCl; repetir una vez. </li><li> Resuspender el pellet en 100 l de NaCl al 0,85% estéril. </li><li> Individualmente detectar 10 l de la cultura lavado para cada cepa en una placa de agar simple TY. Estos puntos son puntos de control. </li><li> Añadir 40 l de cada cepa de E. excluyendo el coli DH5a 12 que contiene pJC2 en un tubo estéril, mezclar, y el punto 120 l de esta mezcla sobre una placa de agar simple TY. Este punto es el control sin la integrasa negativo. </li><li> Añadir 40 l de cada cepa en un t estérilube, mezcla, y el punto 160 l de esta mezcla sobre una placa de agar simple TY. Este lugar es el lugar de apareamiento IMCE. </li><li> Permita que los puntos que se seque en una campana de flujo laminar. </li><li> Sello placas y se incuba a 30 ° C durante la noche. </li></ol> 3. Aislamiento de Trans-integrantes <ol><li> Manchas de imagen unidimensional de control y terreno IMCE sobre discos de agar TY complementadas con 200 ug / ml de estreptomicina (SmUW227 se basa en Rm1021 que lleva una mutación que confiere resistencia de alto nivel a la estreptomicina 14) y gentamicina 30 mg / ml. </li><li> Para el cálculo de la eficiencia IMCE, resuspender aproximadamente ¼ de la mancha de apareamiento IMCE en 500 l estéril al 0,85% de NaCl. Hacer 10 veces diluciones seriadas de 10 -7. Diluciones de placa 10 -2 a través de 10 -5 en placas de agar TY complementadas con 200 ug / ml de estreptomicina y 30 ug / ml de gentamicina, para seleccionar para trans-integrantes. Diluciones Plate 10 -3 through 10 -7 en placas de agar TY complementadas con 200 ug / ml de estreptomicina, para seleccionar para ambos trans integrantes y receptores potenciales, es decir, los receptores totales. </li><li> Para el aislamiento de markerless trans integrantes resuspender aproximadamente un cuarto del punto de acoplamiento IMCE en 500 l de estéril al 0,85% de NaCl. Hacer 10 veces diluciones seriadas hasta 10 -6. Placas 10 -4 a 10 -6 sobre cuatro placas de agar TY complementadas con 200 ug / ml de estreptomicina y 200 ug / ml de X-gluc diluciones. </li><li> Incubar las placas a 30 ° C durante 3 días. </li><li> Ver RFP trans-integrantes bajo la luz verde (525 nm) ya través de un filtro rojo (&gt; 610 nm) 15. </li><li> Calcular la eficiencia IMCE por ciento de UFC de trans-integrantes en comparación con el UFC de los beneficiarios totales. Aproximadamente la mitad de trans-integrantes se han sometido a cambio de cassette de verdad haciéndolos blanco y espectinomicina sensible. </li><li> Para encontrar los markerless trans integrantes (waquí el vector de donantes no contiene Gm r como en pJH110) la pantalla para una colonia de color blanco (un prolongado período de incubación, 1-2 días adicionales a temperatura ambiente, mejorará la diferenciación de las colonias a través de X-gluc, esto es necesario en S. meliloti SmUW227), confirman la sensibilidad de la colonia espectinomicina por el análisis en una placa de agar TY sin antibióticos y una placa de agar TY que contenía 100 mg / ml de espectinomicina. </li></ol> 4. Los resultados representativos Después de tres días de incubación en TY complementadas con estreptomicina y gentamicina las rayas de control no debe tener ningún crecimiento. La raya IMCE debe tener crecimiento confluente en la raya de cabeza y muchas colonias en la franja segundo, como se ve en la Figura 2. La eficiencia de trans-integración, expresada como el porcentaje de trans-integrantes a los destinatarios totales, debe estar en el intervalo de 0,5%. Aproximadamente la mitad de trans-integrantes será espectinomicina sensible y blancomostrando que han sido objeto de cambio de cassette de verdad. Trans-integrantes que contienen el cassette probador rfp donante de pJH110 debe mostrar fluorescencia de RFP discernible cuando se observa bajo la luz verde (525 nm) ya través de un filtro rojo (&gt; 610 nm) 15. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="Figura 1"/> . Figura 1 ilustración mezcla de conjugación: Ayudado por la expresión de los genes de transferencia de pRK600, todos los plásmidos se transfieren al azar de célula a célula. Esta transferencia de los resultados en la creación de trans-integrantes de la mezcla, a través de adquisición del sistema LP-deformación de los dos plásmidos necesarios para IMCE, la integrasa (int) pJC2 plásmido auxiliar y el pJH110 plásmido donante. El cassette de los donantes del plásmido donante no replicante (pJH110) se intercambia a través de la actividad de la integrasa ΦC31 con los marcadores de la LP-cassette en el cromosoma, lo que resulta en la pérdida de la LP-marcadores (Sp.r y uidA) y el mantenimiento de la casete de donante (RFP y r gm) en la resultante trans-integrante. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="Figura 2"/> Figura 2. A: B placa apareamiento lugar en una placa no selectivo TY mostrando mezclas secas de células en la superficie de agar. Manchas de apareamiento en bandas sobre estreptomicina-gentamicina-X-gluc placa, desde la parte superior izquierda hacia la derecha sin control de la integrasa, IMCE en S. meliloti, E. coli DH5a que contiene el control pJC2, E. E. DH5a contiene pJH110 de control, E. E. MT616 de control, y S. . meliloti UW227 de control C: 10 -2 dilución de la resuspensión de apareamiento puesto en TY estreptomicina-X-gluc agar D:. 10 -2 dilución de la resuspensión de apareamiento puesto en TY estreptomicina-gentamicina-X-gluc agar (colonias azules son recombinantes individuales, colonias de color blanco han sido objeto de cierto. de cambio de cassette) E: 10 -2 dilución de la resuspensión de apareamiento puesto en TY estreptomicina-X-gluc agar que muestra la falta de fluorescencia F:. 10 -2 dilución de la resuspensión de apareamiento puesto en TY estreptomicina-gentamicina-X-gluc agar que muestra a dos niveles de la fluorescencia (más brillantes corresponden a las colonias de colonias de color azul y tienen una mayor expresión de rfp presumiblemente debido a la promotora de lectura-aunque a partir de la secuencia del vector, donde las colonias de haber sufrido cierto-cassette de cambio contienen RFP con sólo su promotor inmediato sin leer a través del promotor lac en el vector, que está ausente.).

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C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+site-specific+integration+of+bacteriophage+DNA+catalyzed+by+a+recombinase+of+the+resolvase&#47;invertase+family.">In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase&#47;invertase family.</a> Proc. Natl. Acad. Sci. 95, 5505-5510 (1998).</li><li>Jones, J. D., Gutterson, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+mobilizable+cosmid+vector,+pRK7813,+and+its+use+in+a+rapid+method+for+marker+exchange+in+Pseudomonas+fluorescens+strain+HV37a.">An efficient mobilizable cosmid vector, pRK7813, and its use in a rapid method for marker exchange in Pseudomonas fluorescens strain HV37a.</a> Gene. 61, 299-306 (1987).</li><li>Beringer, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=R+Factor+transfer+in+Rhizobium+leguminosarum.">R Factor transfer in Rhizobium leguminosarum.</a> J. Gen. Microbiol. 84, 188-198 (1974).</li><li>Lennox, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transduction+of+linked+genetic+characters+of+the+host+by+bacteriophage+P1.">Transduction of linked genetic characters of the host by bacteriophage P1.</a> Virology. 1, 190-206 (1955).</li><li>Hanahan, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Studies+on+transformation+of+Escherichia+coli+with+plasmids.">Studies on transformation of Escherichia coli with plasmids.</a> J. Mol. Biol. 166, 557-580 (1983).</li><li>Charles, T. C., Finan, T. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+map+of+Rhizobium+meliloti+megaplasmid+pRmeSU47b.">Genetic map of Rhizobium meliloti megaplasmid pRmeSU47b.</a> J. Bacteriol. 172, 2469-2476 (1990).</li><li>Leigh, J. A., Signer, E. R., Walker, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exopolysaccharide-deficient+mutants+of+Rhizobium+meliloti+that+form+ineffective+nodules.">Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules.</a> Proc. Natl. Acad. Sci. U.S.A. 82, 6231-6235 (1985).</li><li>Heil, J. R., Nordeste, R. F., Charles, T. 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No hay conflictos de interés declarado.

La técnica IMCE permite la integración eficiente de un cassette de ADN única attB flanqueado en el LP-locus de una cepa previamente diseñado. Una vez que la construcción deseada se clona en lugar de RFP para crear el casete donante, la técnica no requiere la purificación posterior del ADN y la transformación, lo que es muy robusto. Es crítico que los controles apropiados de crecimiento se incluyen, para estar seguro de la resistencia a los antibióticos se debe a la creación de trans-integran...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> Estreptomicina </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> La espectinomicina </td><td> Bioshop Canada Inc. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamicina </td><td> Bioshop Canada Inc. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Canada Inc. </td><td> CLR201 </td><td></td></tr><tr><td> La tetraciclina </td><td> Bioshop Canada Inc. </td><td> TET701 </td><td></td></tr><tr><td> Kanamicina </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Agar bacteriológico de grado </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Triptona </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Extracto de levadura </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Cloruro de Sodio </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Cloruro de calcio </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Gold Biotechnology Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli cepa MT616 </td><td> Disponible bajo pedido </td><td></td><td> También se usa fuera de nuestro laboratorio </td></tr><tr><td> E. coli cepa pJC2 </td><td> En la casa, disponible bajo pedido </td><td></td><td></td></tr><tr><td> E. coli cepa pJH110 </td><td> En la casa, a disposición por request </td><td></td><td></td></tr><tr><td> SmUW227 cepa </td><td> En la casa, disponible bajo pedido </td><td></td><td></td></tr></tbody></table>

site-specific bacterial chromosome engineering: &#934;c31 integrase mediated cassette exchange (imce)

El cromosoma bacteriano se puede utilizar para mantener establemente ADN extraño en el intervalo de mega-base 1. La integración en el cromosoma elude cuestiones tales como la replicación del plásmido, la estabilidad del plásmido, la incompatibilidad del plásmido, y la varianza plásmido número de copias. Este método usa la integrasa sitio-específico del fago Streptomyces (Φ) C31 2,3. La integrasa ΦC31 cataliza la recombinación directa entre dos sitios de ADN específicas: attB y attP (34 y 39 pb, respectivamente) 4. Esta recombinación es estable y no se volverá 5. Una &quot;pista de aterrizaje&quot; (LP) consiste en la secuencia de un gen de resistencia a la espectinomicina, aadA (SPR), y E. de la coli ß-glucuronidasa gen (uidA) flanqueado por sitios attP se ha integrado en los cromosomas de Sinorhizobium meliloti, anthropi Ochrobactrum y Agrobacterium tumefaciens en una región intergénica, el ampC locus, y el locus tetA, respectivamente. S. meliloti se utiliza en este protocolo. Vectores que contienen sitios donantes movilizable attB flanquean una embutidora rojo proteína fluorescente (RFP) de genes y un gen de resistencia a antibióticos se han construido. En este ejemplo la gentamicina pJH110 plásmido resistente se utiliza. El gen rfp 6 puede ser reemplazado con un constructo deseado utilizando Sph I y Pst I. Alternativamente una construcción sintética flanqueado por sitios attB puede ser sub-clonado en un vector movilizable como pK19mob 7. La expresión del gen de la integrasa ΦC31 (clonado a partir de pHS62 8) es impulsado por el promotor lac, en una amplia gama de huéspedes movilizable plásmido pRK7813 9. Un protocolo de apareamiento tetraparental se utiliza para transferir el cassette en la cepa donante LP reemplazando así los marcadores en la secuencia de LP con el cassette de donantes. Estas células son trans-integrantes. Trans-integrantes se forman con una eficiencia típica de 0,5%. Trans-integrantes se encuentran típicamente en los primeros 500-1,000 colonias seleccionadas por la sensibilidad a antibióticos o cribado azul-blanco, utilizando 5-bromo-4-cloro-3-indolil-beta-D-glucurónico (X-gluc). Este protocolo contiene los procedimientos de apareamiento y la selección para la creación y aislando trans integrantes.

Watch this Scientific Journal Video about Site-specific Bacterial Chromosome Engineering: Î¦C31 Integrase Mediated Cassette Exchange IMCE at JoVE.com

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

Un método rápido y eficiente para integrar ADN ajeno de interés en cepas aceptor pre-hechas, llamadas cepas pista de aterrizaje, se describe. El método permite específica del sitio de integración de un cassette de ADN en el locus de aterrizaje almohadilla de ingeniería de una cepa dada, a través de la conjugación y expresión de la integrasa ΦC31.

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 ...

site-specific-bacterial-chromosome-engineering-c31-integrase-mediated

Research

JoVE Journal

Bioengineering

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

15.5K vistas.  University of Waterloo. Un método rápido y eficiente para integrar ADN ajeno de interés en cepas aceptor pre-hechas, llamadas cepas pista de aterrizaje, se describe. El método permite específica del sitio de integración de un cassette de ADN en el locus de aterrizaje almohadilla de ingeniería de una cepa dada, a través de la conjugación y expresión de la integrasa ΦC31.

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

Procedure for Lung Engineering

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths annually; chronic obstructive pulmonary disease is currently the fourth leading cause of death in the United States1. Contributing to this mortality is the fact that lungs do not generally repair or regenerate beyond the microscopic, cellular level. Therefore, lung tissue that is damaged by degeneration or infection, or lung tissue that is surgically resected is not functionally replaced in vivo. To explore whether lung tissue can be generated in vitro, we treated lungs from adult rats using a procedure that removes cellular components to produce an acellular lung extracellular matrix scaffold. This scaffold retains the hierarchical branching structures of airways and vasculature, as well as a largely intact basement membrane, which comprises collagen IV, laminin, and fibronectin. The scaffold is mounted in a bioreactor designed to mimic critical aspects of lung physiology, such as negative pressure ventilation and pulsatile vascular perfusion. By culturing pulmonary epithelium and vascular endothelium within the bioreactor-mounted scaffold, we are able to generate lung tissue that is phenotypically comparable to native lung tissue and that is able to participate in gas exchange for short time intervals (45-120 minutes). These results are encouraging, and suggest that repopulation of lung matrix is a viable strategy for lung regeneration. This possibility presents an opportunity not only to work toward increasing the supply of lung tissue for transplantation, but also to study respiratory cell and molecular biology in vitro for longer time periods and in a more accurate microenvironment than has previously been possible.

We have developed a decellularized lung extracellular matrix and novel biomimetic bioreactor that can be used to generate functional lung tissue. By seeding cells into the matrix and culturing in the bioreactor, we generate tissue that demonstrates effective gas exchange when transplanted in vivo for short periods of time.

Procedimiento para la Ingeniería de pulmón

Lung tissue, including lung cancer and chronic lung diseases such as chronic obstructive pulmonary disease, cumulatively account for some 280,000 deaths ...

Investigación

Bioingeniería

High efficiency, Site-specific Transfection of Adherent Cells with siRNA Using Microelectrode Arrays (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 silencing specific genes1-8 for functional genomics and therapeutics. A major challenge involved in RNAi based studies is the delivery of RNAi agents to targeted cells. Traditional non-viral delivery techniques, such as bulk electroporation and chemical transfection methods often lack the necessary spatial control over delivery and afford poor transfection efficiencies9-12. Recent advances in chemical transfection methods such as cationic lipids, cationic polymers and nanoparticles have resulted in highly enhanced transfection efficiencies13. However, these techniques still fail to offer precise spatial control over delivery that can immensely benefit miniaturized high-throughput technologies, single cell studies and investigation of cell-cell interactions. 
Recent technological advances in gene delivery have enabled high-throughput transfection of adherent cells14-23, a majority of which use microscale electroporation. Microscale electroporation offers precise spatio-temporal control over delivery (up to single cells) and has been shown to achieve high efficiencies19, 24-26. Additionally, electroporation based approaches do not require a prolonged period of incubation (typically 4 hours) with siRNA and DNA complexes as necessary in chemical based transfection methods and lead to direct entry of naked siRNA and DNA molecules into the cell cytoplasm. As a consequence gene expression can be achieved as early as six hours after transfection27. Our lab has previously demonstrated the use of microelectrode arrays (MEA) for site-specific transfection in adherent mammalian cell cultures17-19. In the MEA based approach, delivery of genetic payload is achieved via localized micro-scale electroporation of cells. An application of electric pulse to selected electrodes generates local electric field that leads to electroporation of cells present in the region of the stimulated electrodes. The independent control of the micro-electrodes provides spatial and temporal control over transfection and also enables multiple transfection based experiments to be performed on the same culture increasing the experimental throughput and reducing culture-to-culture variability. 
Here we describe the experimental setup and the protocol for targeted transfection of adherent HeLa cells with a fluorescently tagged scrambled sequence siRNA using electroporation. The same protocol can also be used for transfection of plasmid vectors. Additionally, the protocol described here can be easily extended to a variety of mammalian cell lines with minor modifications. Commercial availability of MEAs with both pre-defined and custom electrode patterns make this technique accessible to most research labs with basic cell culture equipment.

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

The article details the protocol for site-specific transfection of scrambled sequence of siRNA in an adherent mammalian cell culture using a microelectrode array (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 ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Capilar Fuerza Litografía Cardiaca de Ingeniería de Tejidos

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global reorganization and realignment of the gel microstructure. This process proceeds in a complex manner that is dependent in part on the interplay between the location of the cells, the geometry of the gel, and the mechanical constraints on the gel. To better understand how these variables produce global fiber alignment patterns, we use time-lapse differential interference contrast (DIC) microscopy coupled with an environmentally controlled bioreactor to observe the compaction process between geometrically spaced explants (clusters of fibroblasts). The images are then analyzed with a custom image processing algorithm to obtain maps of the strain. The information obtained from this technique can be used to probe the mechanobiology of various cell-matrix interactions, which has important implications for understanding processes in wound healing, disease development, and tissue engineering applications.

Time-lapse microscopy and image processing techniques were used to observe and analyze fibroblast-mediated gel compaction and fibrin fiber realignment in an environmentally controlled bioreactor over a 48 hr&#160;period.

Observación Y Cuantificación De Fibroblastos Mediada Por Fibrina Gel Compactamiento

Cells embedded in collagen and fibrin gels attach and exert traction forces on the fibers of the gel. These forces can lead to local and global ...

Synthesis of an Intein-mediated Artificial Protein Hydrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of this hydrogel are two protein block-copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. A highly hydrophilic random coil is inserted into one of the block-copolymers for water retention. Mixing of the two protein block copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit with crosslinkers at either end that rapidly self-assembles into a hydrogel. This hydrogel is very stable under both acidic and basic conditions, at temperatures up to 50 &#176;C, and in organic solvents. The hydrogel rapidly reforms after shear-induced rupture. Incorporation of a &#34;docking station peptide&#34; into the hydrogel building block enables convenient incorporation of &#34;docking protein&#34;-tagged target proteins. The hydrogel is compatible with tissue culture growth media, supports the diffusion of 20 kDa molecules, and enables the immobilization of bioactive globular proteins. The application of the intein-mediated protein hydrogel as an organic-solvent-compatible biocatalyst was demonstrated by encapsulating the horseradish peroxidase enzyme and corroborating its activity.

We present the synthesis of a split-intein-mediated protein hydrogel. The building blocks of this hydrogel are two protein copolymers each containing a subunit of a trimeric protein that serves as a crosslinker and one half of a split intein. Mixing of the two protein copolymers triggers an intein trans-splicing reaction, yielding a polypeptide unit that self-assembles into a hydrogel. This hydrogel is highly pH- and temperature-stable, compatible with organic solvents, and easily incorporates functional globular proteins.

Síntesis de una proteína artificial mediada Intein hidrogel

We present the synthesis of a highly stable protein hydrogel mediated by a split-intein-catalyzed protein trans-splicing reaction. The building blocks of ...

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose and short sisal fibers are dispersed into a water suspension containing bacterial cellulose. The fiber and nanocellulose suspension is then filtered (using vacuum or gravity) and the wet filter cake pressed to squeeze out any excess water, followed by a drying step. This will result in the hornification of the bacterial cellulose network, holding the loose natural fibers together.
Our method is specially suited for the manufacturing of rigid and robust preforms of hydrophilic fibers. The porous and hydrophilic nature of such fibers results in significant water uptake, drawing in the bacterial cellulose dispersed in the suspension. The bacterial cellulose will then be filtered against the surface of these fibers, forming a bacterial cellulose coating. When the loose fiber-bacterial cellulose suspension is filtered and dried, the adjacent bacterial cellulose forms a network and hornified to hold the otherwise loose fibers together.
The introduction of bacterial cellulose into the preform resulted in a significant increase of the mechanical properties of the fiber preforms. This can be attributed to the high stiffness and strength of the bacterial cellulose network. With this preform, renewable high performance hierarchical composites can also be manufactured by using conventional composite production methods, such as resin film infusion (RFI) or resin transfer molding (RTM). Here, we also describe the manufacturing of renewable hierarchical composites using double bag vacuum assisted resin infusion.

We present a novel method of manufacturing rigid and robust short natural fiber preforms using a papermaking process. Bacterial cellulose acts simultaneously as the binder for the loose fibers and provides rigidity to the fiber preforms. These preforms can be infused with a resin to produce truly green hierarchical composites.

Fabricación De robustos Fibra Natural Preformas Utilizando bacteriana celulosa como Carpeta

A novel method of manufacturing rigid and robust natural fiber preforms is presented here. This method is based on a papermaking process, whereby loose ...

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea (ureolysis) in naturally occurring environments through secretion of an enzyme urease. This process of ureolysis, through a chain of chemical reactions, leads to the formation of calcium carbonate precipitates. This is known as Microbiologically Induced Calcite Precipitation (MICP). The proper culture protocols for MICP are detailed here. Finally, visualization experiments under different modes of microscopy were performed to understand various aspects of the precipitation process. Techniques like optical microscopy, Scanning Electron Microscopy (SEM) and X-Ray Photo-electron Spectroscopy (XPS)&#160;were employed to chemically characterize the end-product. Further, the ability of these precipitates to clog pores inside a natural porous medium was demonstrated through a qualitative experiment where sponge bars were used to mimic a pore-network with a range of length scales. A sponge bar dipped in the culture medium containing the bacterial cells hardens due to the clogging of its pores resulting from the continuous process of chemical precipitation. This hardened sponge bar exhibits superior strength when compared to a control sponge bar which becomes compressed and squeezed under the action of an applied external load, while the hardened bar is able to support the same weight with little deformation.

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

Protocols for microbiologically induced calcite precipitation (MICP) using the bacterium Sporosarcina pasteurii are presented here. The precipitated calcium carbonate was characterized through optical microscopy and scanning electron microscopy (SEM). It is also shown that exposure to MICP increases the compressive strength of sponge.

Inducida microbiológicamente Calcita precipitación mediada por<em&gt; Sporosarcina pasteurii</em

The particular bacterium under investigation here (S. pasteurii) is unique in its ability, under the right conditions, to induce the hydrolysis of urea ...

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and trade one defect for another. Tissue engineering holds the promise to address this increasing demand. However, most tissue engineering strategies fail to generate stable and functional tissue substitutes because of poor vascularization. This paper focuses on an in vivo tissue engineering chamber model of intrinsic vascularization where a perfused artery and a vein either as an arteriovenous loop or a flow-through pedicle configuration is directed inside a protected hollow chamber. In this chamber-based system angiogenic sprouting occurs from the arteriovenous vessels and this system attracts ischemic and inflammatory driven endogenous cell migration which gradually fills the chamber space with fibro-vascular tissue. Exogenous cell/matrix implantation at the time of chamber construction enhances cell survival and determines specificity of the engineered tissues which develop. Our studies have shown that this chamber model can successfully generate different tissues such as fat, cardiac muscle, liver and others. However, modifications and refinements are required to ensure target tissue formation is consistent and reproducible. This article describes a standardized protocol for the fabrication of two different vascularized tissue engineering chamber models in vivo.

Tissue Engineering by Intrinsic Vascularization in an In Vivo Tissue Engineering Chamber

This is a guideline for constructing in vivo vascularized tissue using a microsurgical arteriovenous loop or a flow-through pedicle configuration inside a tissue engineering chamber. The vascularized tissues generated can be employed for organ regeneration and replacement of tissue defects, as well as for drug testing and disease modeling.

Ingeniería de tejidos por intrínseca Vascularización en una<em&gt; En Vivo</em&gt; Cámara Ingeniería de Tejidos

In reconstructive surgery, there is a clinical need for an alternative to the current methods of autologous reconstruction which are complex, costly and ...

Microfluidic Buffer Exchange for Interference-free Micro/Nanoparticle Cell Engineering

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic properties, enable bio imaging and control cell phenotype. A critical yet inadequately addressed issue is the significant number of particles that remain unbound after cell labeling which cannot be readily removed by conventional centrifugation. This leads to an increase in bio imaging background noise and can impart transformative effects onto neighboring non-target cells. In this protocol, we present an inertial microfluidics-based buffer exchange strategy termed as Dean Flow Fractionation (DFF) to efficiently separate labeled cells from free NPs in a high throughput manner. The developed spiral microdevice facilitates continuous collection (&#62;90% cell recovery) of purified cells (THP-1 and MSCs) suspended in new buffer solution, while achieving &#62;95% depletion of unbound fluorescent dye or dye-loaded NPs (silica or PLGA). This single-step, size-based cell purification strategy enables high cell processing throughput (106 cells/min) and is highly useful for large-volume cell purification of micro/nanoparticle engineered cells to achieve interference-free clinical application.

This protocol describes the use of an inertial microfluidics-based buffer exchange strategy to purify micro/nanoparticle engineered cells with efficient depletion of unbound particles.

Microfluidos intercambio de tampón para la Micro / célula de nanopartículas Ingeniería sin interferencias

Engineering cells with active-ingredient-loaded micro/nanoparticles (NPs) is becoming an increasingly popular method to enhance native therapeutic ...

Light-mediated Formation and Patterning of Hydrogels for Cell Culture Applications

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In particular, light-mediated click reactions, such as photoinitiated thiol&#8722;ene and thiol&#8722;yne reactions, afford spatiotemporal control over material properties and allow the design of systems with a high degree of user-directed property control. Fabrication and modification of hydrogel-based biomaterials using the precision afforded by light and the versatility offered by these thiol&#8722;X photoclick chemistries are of growing interest, particularly for the culture of cells within well-defined, biomimetic microenvironments. Here, we describe methods for the photoencapsulation of cells and subsequent photopatterning of biochemical cues within hydrogel matrices using versatile and modular building blocks polymerized by a thiol&#8722;ene photoclick reaction. Specifically, an approach is presented for constructing hydrogels from allyloxycarbonyl (Alloc)-functionalized peptide crosslinks and pendant peptide moieties and thiol-functionalized poly(ethylene glycol) (PEG) that rapidly polymerize in the presence of lithium acylphosphinate photoinitiator and cytocompatible doses of long wavelength ultraviolet (UV) light. Facile techniques to visualize photopatterning and quantify the concentration of peptides added are described. Additionally, methods are established for encapsulating cells, specifically human mesenchymal stem cells, and determining their viability and activity. While the formation and initial patterning of thiol-alloc hydrogels are shown here, these techniques broadly may be applied to a number of other light and radical-initiated material systems (e.g., thiol-norbornene, thiol-acrylate) to generate patterned substrates.

We describe a sequential process for light-mediated formation and subsequent biochemical patterning of synthetic hydrogel matrices for three-dimensional cell culture applications. The construction and modification of hydrogels with cytocompatible photoclick chemistry is demonstrated. Additionally, facile techniques to quantify and observe patterns and determine cell viability within these hydrogels are presented.

Formación luz mediada y el patrón de hidrogeles para aplicaciones de cultivo celular

Click chemistries have been investigated for use in numerous biomaterials applications, including drug delivery, tissue engineering, and cell culture. In ...