Este trabajo fue apoyado por la NSF RUI Becas MCB-0950372 y DMR-1105310 titulada &quot;Caracterización molecular de Black Widow sedas de araña y comportamiento mecánico de araña Sedas pegamento&quot;, respectivamente.

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A., Bittencourt, D., Rech, E. L., Brooks, A., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+protocol+for+the+production+of+recombinant+spider+silk-like+proteins+for+artificial+fiber+spinning.">A protocol for the production of recombinant spider silk-like proteins for artificial fiber spinning.</a> Nat. Protoc. 4, 341-355 (2009).</li><li>Gnesa, E., Hsia, Y., Yarger, J. L., Weber, W., Lin-Cereghino, J., Lin-Cereghino, G., Tang, S., Agari, K., Vierra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conserved+C-Terminal+Domain+of+Spider+Tubuliform+Spidroin+1+Contributes+to+Extensibility+in+Synthetic+Fibers.">Conserved C-Terminal Domain of Spider Tubuliform Spidroin 1 Contributes to Extensibility in Synthetic Fibers.</a> Biomacromolecules. , (2011).</li><li>Hayashi, C. Y., Shipley, N. H., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypotheses+that+correlate+the+sequence,+structure,+and+mechanical+properties+of+spider+silk+proteins.">Hypotheses that correlate the sequence, structure, and mechanical properties of spider silk proteins.</a> Int. J. Biol. Macromol. 24, 271-275 (1999).</li><li>Xu, M., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+a+protein+superfiber:+Spider+Dragline+Silk.">Structure of a protein superfiber: Spider Dragline Silk.</a> Proc. Natl. Acad. Sci. 87, 7120-7124 (1990).</li><li>Hayashi, C. Y., Blackledge, T. A., Lewis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+and+mechanical+characterization+of+aciniform+silk:+uniformity+of+iterated+sequence+modules+in+a+novel+member+of+the+spider+silk+fibroin+gene+family.">Molecular and mechanical characterization of aciniform silk: uniformity of iterated sequence modules in a novel member of the spider silk fibroin gene family.</a> Mol. Biol. Evol. 21, 1950-1959 (2004).</li><li>Lazaris, A., Arcidiacono, S., Huang, Y., Zhou, J. F., Duguay, F., Chretien, N., Welsh, E. A., Soares, J. W., Karatzas, C. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+silk+fibers+spun+from+soluble+recombinant+silk+produced+in+mammalian+cells.">Spider silk fibers spun from soluble recombinant silk produced in mammalian cells.</a> Science. 295, 472-476 (2002).</li><li>Arcidiacono, S., Mello, C., Kaplan, D., Cheley, S., Bayley, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Purification+and+characterization+of+recombinant+spider+silk+expressed+in+Escherichia+coli.">Purification and characterization of recombinant spider silk expressed in Escherichia coli.</a> Appl. Microbiol. Biotechnol. 49, 31-38 (1998).</li><li>Menassa, R., Zhu, H., Karatzas, C. N., Lazaris, A., Richman, A., Brandle, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+dragline+silk+proteins+in+transgenic+tobacco+leaves:+accumulation+and+field+production.">Spider dragline silk proteins in transgenic tobacco leaves: accumulation and field production.</a> Plant Biotechnology Journal. 2, 431-438 (2004).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nat. Biotechnol. 19, 573-577 (2001).</li><li>An, B., Hinman, M. B., Holland, G. P., Yarger, J. L., Lewis, R. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducing+beta-sheets+formation+in+synthetic+spider+silk+fibers+by+aqueous+post-spin+stretching.">Inducing beta-sheets formation in synthetic spider silk fibers by aqueous post-spin stretching.</a> Biomacromolecules. 12, 2375-2381 (2011).</li><li>Elices, M., Guinea, G. V., Plaza, G. R., Karatzas, C., Riekel, C., Agullo-Rueda, F., Daza, R., Perez-Rigueiro, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioinspired Fibers Follow the Track of Natural Spider Silk. Macromolecules. 44, 1166-1176 (2011).</li><li>Scheller, J., Guhrs, K. H., Grosse, F., Conrad, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+spider+silk+proteins+in+tobacco+and+potato.">Production of spider silk proteins in tobacco and potato.</a> Nature Biotechnology. 19, (2001).</li><li>Kojic, N., Kojic, M., Gudlavalleti, S., McKinley, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solvent+removal+during+synthetic+and+Nephila+fiber+spinning.">Solvent removal during synthetic and Nephila fiber spinning.</a> Biomacromolecules. 5, 1698-1707 (2004).</li><li>Jeffery, F., La Mattina, C., Tuton-Blasingame, T., Hsia, Y., Gnesa, E., Zhao, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microdissection+of+Black+Widow+Spider+Silk-producing+Glands.">Microdissection of Black Widow Spider Silk-producing Glands.</a> J. Vis. Exp. (47), e2382 (2011).</li><li>Blasingame, E., Tuton-Blasingame, T., Larkin, L., Falick, A. M., Zhao, L., Fong, J., Vaidyanathan, V., Visperas, A., Geurts, P., Hu, X., La Mattina, C., Vierra, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pyriform+spidroin+1,+a+novel+member+of+the+silk+gene+family+that+anchors+dragline+silk+fibers+in+attachment+discs+of+the+black+widow+spider,+Latrodectus+hesperus.">Pyriform spidroin 1, a novel member of the silk gene family that anchors dragline silk fibers in attachment discs of the black widow spider, Latrodectus hesperus.</a> J. Biol. Chem. 284, 29097-29108 (2009).</li><li>La Mattina, C., Reza, R., Hu, X., Falick, A. M., Vasanthavada, K., McNary, S., Yee, R., Vierra, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+minor+ampullate+silk+proteins+are+constituents+of+prey+wrapping+silk+in+the+cob+weaver+Latrodectus+hesperus.">Spider minor ampullate silk proteins are constituents of prey wrapping silk in the cob weaver Latrodectus hesperus.</a> Biochemistry. 47, 4692-4700 (2008).</li><li>Hsia, Y., Gnesa, E., Jeffery, F., Tang, S., Vierra, C., Cuppoletti, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spider+Silk+Composites+and+Applications.">Spider Silk Composites and Applications.</a> Metal, Ceramic and Polymeric Composites for Various Uses. 2, 303-324 (2011).</li></ol>

No hay conflictos de interés declarado.

Las fibras sintéticas hiladas a partir de esta metodología son mecánicamente en el mismo orden de magnitud en comparación con las fibras naturales. Al disminuir la cantidad de errores humanos mediante la mecanización de la cola de impresión y los procesos posteriores de giro del sorteo, la variación entre muestras experimentales son más controlado y reducido en gran medida. Nuestra metodología ofrece la posibilidad de investigar las propiedades mecánicas de las fibras de otros que ...

La seda de araña tiene extraordinarias propiedades mecánicas que lleva a cabo a cabo varios materiales hechos por el hombre, incluyendo acero de alta resistencia, kevlar y nylon. 1 Las arañas tejen por lo menos 6-7 diferentes tipos de fibras que presentan diversas propiedades mecánicas, cada uno diseñado con cantidades variables de resistencia a la tracción y capacidad de ampliación para llevar a cabo tareas específicas de biológicos. 2 Los investigadores científicos están buscando rápidamente el uso de sedas de araña como biomateriales de próxima generación, debido a sus excelentes propiedades mecánicas, de su biocompatibilidad, y su carácter no tóxico y material verde. 3,4 Debido a la caníbal y la naturaleza venenosa de los arácnidos, la cosecha sedas de araña a través de la agricultura no es una estrategia práctica para satisfacer las demandas necesarias para la fabricación a escala industrial. Por lo tanto, los científicos han recurrido a la producción de proteínas recombinantes en seda organismos transgénicos, junto con in vitro hilado de fibras sintéticas de lasE proteínas purificadas. 8.5 Expresión de larga duración en proteínas recombinantes de seda de araña ha sido técnicamente difícil, dadas las propiedades intrínsecas de sus secuencias genéticas, que incluyen su naturaleza altamente repetitivo y longitudes físicas (&gt; 15 kb), GC-rica de contenido y parcial alanina y glicina uso de codones. 9.11 Hasta la fecha, la mayoría de los laboratorios se han centrado en la expresión de formas truncadas de las principales proteínas de la seda ampullate MaSp1 o MASP2 usando secuencias parciales de ADNc o genes sintéticos. 12-15 Spinning sintética las sedas de araña es un proceso difícil que requiere el dominio y el conocimiento en varias disciplinas científicas, y las complejidades del proceso de hilado no se han revelado al público en general por la representación de vídeo. De hecho, sólo un puñado de laboratorios en todo el mundo tienen la experiencia para expresar el ADNc de seda de araña, purificar las proteínas de la seda, hacer girar las fibras sintéticas y llevar a cabo después de la vuelta empate, y, finalmente, probar sus propiedades de biomateriales. 8,16,17 Diferentes enfoques para la hilatura de fibras sintéticas han abarcado hilado en húmedo y seco, así como los métodos de electrospinning 16,18,19 Todos los procedimientos tienen una meta en común -. El desarrollo de un protocolo que produce la seda de araña sintética con propiedades mecánicas que rivalizan con hilos naturales a gran escala los procesos de fabricación comerciales. A continuación se describe el procedimiento para generar las sedas de araña artificiales a escala de laboratorio utilizando una metodología de hilatura en húmedo. En comparación con otros métodos de hilado, hilado en húmedo ha producido los resultados más consistentes para el análisis de fibra. Se describen este procedimiento comenzando con la expresión de las proteínas recombinantes en bacterias de seda, seguido por la purificación de ellos y, a continuación se describen los pasos de preparación de proteínas para el hilado, incluyendo una metodología sorteo posterior giro aplicado a &quot;como&quot; fibras hiladas en que los rendimientos de hilos con propiedades de los materiales que se acercan a la calidad de las sedas de araña naturales. Nuestro METODOLOGÍy está diseñado para imitar el proceso natural de hilado de fibras de seda y se basa en gran medida nuestra experiencia de la arquitectura y el funcionamiento de las glándulas productoras de seda de la orbe-y mazorca de tejido de las arañas. 20-22 Además, se concluye con la necesaria pasos para determinar las propiedades del material de las fibras sintéticas utilizando un tensiómetro para trazar curvas tensión-deformación, que permiten a los investigadores a calcular la resistencia última, deformación última, y ​​la tenacidad de las fibras. Por último, pero de gran valor, los aparatos de hilatura, cola, y el dibujo puede estar en casa-construida con piezas disponibles en el mercado, en lugar de la compra de equipos a medida elaborado y costoso.

<table border="1"><tbody><tr><td> Reactivo / Equipo </td><td> Empresa </td><td> Número de catálogo </td><td> Comentarios </td></tr><tr><td> pBAD / TOPO ThioFusion Kit de Expresión </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> Lisis de la célula FastBreak reactivo, 10x </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> Ni-NTA agarosa </td><td> Qiagen </td><td> 30210 </td><td> Incluye instrucciones para tampones </td></tr><tr><td> ProteoSilver kit de tinción de plata </td><td> Sigma-Aldrich </td><td> PROTSIL1-1KT </td><td></td></tr><tr><td> FreeZone liofilizador </td><td> Labconco </td><td> 7960041 </td><td> FreeZone 12plus </td></tr><tr><td> Hexafluoroisopropanol (HFIP) </td><td> Sigma-Aldrich </td><td> 52512 </td><td></td></tr><tr><td> Jeringa </td&gt; <td> Hamilton </td><td> 7657-01 </td><td> 250 L </td></tr><tr><td> Aguja </td><td> Hamilton </td><td> 7780-01 </td><td> Medidor de 26s, extremo romo aguja intercambiable; </td></tr><tr><td> Bomba de jeringa </td><td> Aparato de Harvard </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> Caliper Digital </td><td> Carrera </td><td> CP5906 </td><td> Rango 0-150 mm </td></tr><tr><td> Pinzas de acero inoxidable </td><td> World Precision Instruments </td><td> 501764 </td><td> Mini Dumont # M5S </td></tr><tr><td> Motor </td><td> Naturaleza Molino </td><td> 7090529 </td><td> 12 V CC, 2 rpm de velocidad </td></tr><tr><td> Actuador lineal </td><td> Warner Electric </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC, rango de 6 pulgadas </td></tr><tr><td> Microscopio de disección </td><td> Leica Microsystems </td><td> Leica MZ16 </td><td></td></tr><tr><td> Digital MicroscOpe cámara </td><td> Leica Microsystems </td><td> DFC320 </td><td> Software: Leica Application Suite v2.8.1 </td></tr><tr><td> Vannas tijera </td><td> World Precision Instruments </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> Aurora, la Ciencia </td><td> 310C </td><td> 5N de modo dual del sistema </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

Mientras la sociedad avanza y los recursos se vuelven escasos, cada vez es más importante cultivar las nuevas tecnologías que los biomateriales de ingeniería de última generación con propiedades de alto rendimiento. El desarrollo de estos nuevos materiales estructurales debe ser rápida, rentable y hacer participar a las metodologías de procesamiento y productos respetuosos con el medio ambiente y sostenible. Las arañas tejen una multitud de diferentes tipos de fibras con diversas propiedades mecánicas, que ofrece una rica fuente de material próximos generación de ingeniería para el biomimetismo que rivalizan con los mejores materiales naturales y artificiales. Desde la recogida de grandes cantidades de seda de araña natural es poco práctico, la producción de seda sintética tiene la capacidad de proporcionar a los científicos el acceso a un suministro ilimitado de temas. Por lo tanto, si el proceso de hilatura puede ser simplificado y perfeccionado, fibras artificiales araña tiene el uso potencial de una amplia gama de aplicaciones que van desde chalecos antibalas, sutura quirúrgicas, cuerdas y cordajes, neumáticos, cuerdas para instrumentos musicales, y materiales compuestos para la aviación y la tecnología aeroespacial. Con el fin de avanzar en el proceso de producción de seda sintética y para producir fibras que muestran poca variación en sus propiedades materiales de la vuelta a girar, se desarrolló un protocolo de hilatura en húmedo que se integra la expresión de proteínas recombinantes de seda de araña en las bacterias, la purificación y concentración de las proteínas , seguido por extrusión de fibra y un tratamiento mecánico posterior giro. Esta es la primera representación visual que revela un proceso paso a paso para hacer girar y analizar fibras de seda artificial a escala de laboratorio. También proporciona detalles para minimizar la introducción de variabilidad entre las fibras hiladas a partir de la mezcla de hilatura mismo. En conjunto, estos métodos impulsará el proceso de producción de seda artificial, lo que lleva a las fibras de mayor calidad que superan las sedas de araña naturales.

Watch this Scientific Journal Video about Synthetic Spider Silk Production on a Laboratory Scale at JoVE.com

Synthetic Spider Silk Production on a Laboratory Scale

A pesar de las excelentes propiedades mecánicas y bioquímicas de las sedas de araña, este material no pueden ser cosechadas en grandes cantidades por medios convencionales. A continuación se describe una estrategia eficiente para hacer girar las fibras artificiales de seda de araña, que es un proceso importante para los investigadores que estudian la producción de seda de araña y su uso como la próxima generación de biomateriales.

La producción de seda sintética araña a escala de laboratorio

As society progresses and resources become scarcer, it is becoming increasingly important to cultivate new technologies that engineer next generation ...

synthetic-spider-silk-production-on-a-laboratory-scale

Research

JoVE Journal

Bioengineering

26.6K vistas.  University of the Pacific. A pesar de las excelentes propiedades mecánicas y bioquímicas de las sedas de araña, este material no pueden ser cosechadas en grandes cantidades por medios convencionales. A continuación se describe una estrategia eficiente para hacer girar las fibras artificiales de seda de araña, que es un proceso importante para los investigadores que estudian la producción de seda de araña y su uso como la próxima generación de biomateriales.

Video: Synthetic Spider Silk Production on a Laboratory Scale

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

Filtro de aire dispositivos, incluyendo mallas tejidas de electrospun recombinantes Las proteínas de seda de araña

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Investigación

Bioingeniería

Designing Silk-silk Protein Alloy Materials for Biomedical Applications

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, nanomedicine, tissue regeneration, and drug delivery. Designing materials based on the molecular-scale interactions between these proteins will help generate new multifunctional protein alloy biomaterials with tunable properties. Such alloy material systems also provide advantages in comparison to traditional synthetic polymers due to the materials biodegradability, biocompatibility, and tenability in the body. This article used the protein blends of wild tussah silk (Antheraea pernyi) and domestic mulberry silk (Bombyx mori) as an example to provide useful protocols regarding these topics, including how to predict protein-protein interactions by computational methods, how to produce protein alloy solutions, how to verify alloy systems by thermal analysis, and how to fabricate variable alloy materials including optical materials with diffraction gratings, electric materials with circuits coatings, and pharmaceutical materials for drug release and delivery. These methods can provide important information for designing the next generation multifunctional biomaterials based on different protein alloys.

Blending is an efficient approach to generate biomaterials with a broad range of properties and combined features. By predicting the molecular interactions between different natural silk proteins, new silk-silk protein alloy platforms with tunable mechanical resiliency, electrical response, optical transparency, chemical processability, biodegradability, or thermal stability can be designed.

El diseño de proteínas de seda-seda aleaciones para aplicaciones biomédicas

Fibrous proteins display different sequences and structures that have been used for various applications in biomedical fields such as biosensors, ...

Production and Targeting of Monovalent Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in biology, where clustering and the spatial organization of biomolecules is often the object of study. We describe here a protocol to produce monovalent quantum dots (mQDs) that can be accomplished in most biological research laboratories via a simple mixing of CdSe/ZnS core/shell QDs with phosphorothioate DNA (ptDNA) of defined length. After a single ptDNA strand has wrapped the QD, additional strands are excluded from the surface. Production of mQDs in this manner can be accomplished at small and large scale, with commercial reagents, and in minimal steps. These mQDs can be specifically directed to biological targets by hybridization to a complementary single stranded targeting DNA. We demonstrate the use of these mQDs as imaging probes by labeling SNAP-tagged Notch receptors on live mammalian cells, targeted by mQDs bearing a benzylguanine moiety.

We provide detailed instructions for the preparation of monovalent targeted quantum dots (mQDs) from phosphorothioate DNA of defined length. DNA wrapping occurs in high yield, and therefore, products do not require purification. We demonstrate the use of the SNAP tag to target mQDs to cell-surface receptors for live-cell imaging applications.

Producción y focalización de monovalente Quantum Dots

The multivalent nature of commercial quantum dots (QDs) and the difficulties associated with producing monovalent dots have limited their applications in ...

Cloning and Large-Scale Production of High-Capacity Adenoviral Vectors Based on the Human Adenovirus Type 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high packaging capacity (up to 35 kb), low immunogenicity, and low toxicity. However, for many laboratories the use of HCAdV is hampered by the complicated procedure for vector genome construction and virus production. Here, a detailed protocol for efficient cloning and production of HCAdV based on the plasmid pAdFTC containing the HCAdV genome is described. The construction of HCAdV genomes is based on a cloning vector system utilizing homing endonucleases (I-CeuI and PI-SceI). Any gene of interest of up to 14 kb can be subcloned into the shuttle vector pHM5, which contains a multiple cloning site flanked by I-CeuI and PI-SceI. After I-CeuI and PI-SceI-mediated release of the transgene from the shuttle vector the transgene can be inserted into the HCAdV cloning vector pAdFTC. Because of the large size of the pAdFTC plasmid and the long recognition sites of the used enzymes associated with strong DNA binding, careful handling of the cloning fragments is needed. For virus production, the HCAdV genome is released by NotI digest and transfected into a HEK293 based producer cell line stably expressing Cre recombinase. To provide all adenoviral genes for adenovirus amplification, co-infection with a helper virus containing a packing signal flanked by loxP sites is required. Pre-amplification of the vector is performed in producer cells grown on surfaces and large-scale amplification of the vector is conducted in spinner flasks with producer cells grown in suspension. For virus purification, two ultracentrifugation steps based on cesium chloride gradients are performed followed by dialysis. Here tips, tricks, and shortcuts developed over the past years working with this HCAdV vector system are presented.

A protocol for generation of high-capacity adenoviral vectors lacking all viral coding sequences is presented. Cloning of transgenes contained in the vector genome is based on homing endonucleases. Virus amplification in producer cells grown as adherent cells and in suspension relies on a helper virus providing viral genes in trans.

La clonación y producción a gran escala de alta capacidad vectores adenovirales en base al tipo de adenovirus humano 5

High-capacity adenoviral vectors (HCAdV) devoid of all viral coding sequences represent one of the most advanced gene delivery vectors due to their high ...

Engineered 3D Silk-collagen-based Model of Polarized Neural Tissue

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a deeper comprehension of the neural system scientists aim to simplistically reconstruct the tissue by assembling it in vitro from basic building blocks using a tissue engineering approach. Our group developed a tissue-engineered silk and collagen-based 3D brain-like model resembling the white and gray matter of the cortex. The model consists of silk porous sponge, which is pre-seeded with rat brain-derived neurons, immersed in soft collagen matrix. Polarized neuronal outgrowth and network formation is observed with separate axonal and cell body localization. This compartmental architecture allows for the unique development of niches mimicking native neural tissue, thus enabling research on neuronal network assembly, axonal guidance, cell-cell and cell-matrix interactions and electrical functions.

Insight into the complex actions of the brain requires advanced research tools. Here we demonstrate a novel silk-collagen-based 3D engineered model of neural tissue resembling brain-like architecture. The model can be used to study neuronal network assembly, axonal guidance, cell-cell interactions and electrical activity.

Modelo de polarizado Neural tisular basada Silk-colágeno-Engineered 3D

Despite huge efforts to decipher the anatomy, composition and function of the brain, it remains the least understood organ of the human body. To gain a ...

Power Input Measurements in Stirred Bioreactors at Laboratory Scale

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during rotation. However, the experimental determination of the power input in small-scale vessels is still challenging due to relatively high friction losses inside typically used bushings, bearings and/or shaft seals and the accuracy of commercially available torque meters. Thus, only limited data for small-scale bioreactors, in particular single-use systems, is available in the literature, making comparisons among different single-use systems and their conventional counterparts difficult.
This manuscript provides a protocol on how to measure power inputs in benchtop scale bioreactors over a wide range of turbulence conditions, which can be described by the dimensionless Reynolds number (Re). The aforementioned friction losses are effectively reduced by the use of an air bearing. The procedure on how to set up, conduct and evaluate a torque-based power input measurement, with special focus on cell culture typical agitation conditions with low to moderate turbulence (100 &#60; Re &#60; 2&#183;104), is described in detail. The power input of several multi-use and single-use bioreactors is provided by the dimensionless power number (also called Newton number, P0), which is determined to be in the range of P0 &#8776; 0.3 and P0 &#8776; 4.5 for the maximum Reynolds numbers in the different bioreactors.

The power input in stirred bioreactors can be measured through the torque that acts on the impeller shaft during rotation. This manuscript describes how an air bearing can be used to effectively reduce friction losses observed in mechanical seals and improve the accuracy of power input measurements in small-scale vessels.

Medidas de entrada de potencia en biorreactores agitados a escala de laboratorio

The power input in stirred bioreactors is an important scaling-up parameter and can be measured through the torque that acts on the impeller shaft during ...

Material Formation of Recombinant Spider Silks through Aqueous Solvation using Heat and Pressure

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order to reproduce the remarkable mechanical properties of spider silks, they must be produced in heterologous hosts as spiders are both territorial and cannibalistic. The synthetic analogs of spider silk also tend to be insoluble in aqueous solutions. Thus, a large percentage of research in recombinant spider silks rely upon organic solvents that are detrimental to large scale production of materials. Our group&#8217;s method forces the solvation of these recombinant spider silks into water. Remarkably, when these proteins are prepared using this method of heat and pressure, a wide range of material forms can be prepared from the same solution of recombinant spider silk proteins (rSSp) including: films, fibers, sponge, hydrogel, lyogel, and adhesives. This article demonstrates the production of the solvated rSSp and material forms in a manner that is more easily understood than from written materials and methods alone.

Here, we present a protocol to produce water soluble recombinant spider silk protein solutions and the material forms that can be formed from those solutions.

Formación material de la araña recombinante sedas a través de la Solvación acuosa utilizando calor y presión

Many spiders produce seven types of silks. Six of the silks are fiber in form when produced by the spiders. These fibers are not water soluble. In order ...

Large-Scale, Automated Production of Adipose-Derived Stem Cell Spheroids for 3D Bioprinting

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue recognized as a classical source of mesenchymal stromal/stem cells. Many studies have been published with ASCs for scaffold-based tissue engineering approaches, which mainly explored the behavior of these cells after their seeding on bioactive scaffolds. However, scaffold-free approaches are emerging to engineer tissues in vitro and in vivo, mainly by using spheroids, to overcome the limitations of scaffold-based approaches.
Spheroids are 3D microtissues formed by the self-assembly process. They can better mimic the architecture and microenvironment of native tissues, mainly due to the magnification of cell-to-cell and cell-to-extracellular matrix interactions. Recently, spheroids are mainly being explored as disease models, drug screening studies, and building blocks for 3D bioprinting. However, for 3D bioprinting approaches, numerous spheroids, homogeneous in size and shape, are necessary to biofabricate complex tissue and organ models. In addition, when spheroids are produced automatically, there is little chance for microbiological contamination, increasing the reproducibility of the method.
The large-scale production of spheroids is considered the first mandatory step for developing a biofabrication line, which continues in the 3D bioprinting process and finishes in the full maturation of the tissue construct in bioreactors. However, the number of studies that explored the large-scale ASC spheroid production are still scarce, together with the number of studies that used ASC spheroids as building blocks for 3D bioprinting. Therefore, this article aims to show the large-scale production of ASC spheroids using a non-adhesive micromolded hydrogel technique spreading ASC spheroids as building blocks for 3D bioprinting approaches.

Here, we describe the large-scale production of adipose-derived stromal/stem cell (ASC) spheroids using an automated pipetting system to seed the cell suspension, thus ensuring homogeneity of spheroid size and shape. These ASC spheroids can be used as building blocks for 3D bioprinting approaches.

Producción automatizada a gran escala de esferoides de células madre derivadas de tejido adiposo para bioimpresión 3D

Adipose-derived stromal/stem cells (ASCs) are a subpopulation of cells found in the stromal vascular fraction of human subcutaneous adipose tissue ...

Producción en sistema cerrado de células adherentes basadas en microportadores solubles

Large-Scale Cell Production Based on GMP-Grade Dissolvable Porous Microcarriers

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To address the primary shortcomings of the two-dimensional (2D) planar culturing system, we innovatively developed an automated closed industrial scale cell production (ACISCP) platform based on a GMP-grade, dissolvable, and porous microcarrier for the 3D culture of adherent cells, including human mesenchymal stem/stromal cells (hMSCs), HEK293T cells, and Vero cells. To achieve large-scale expansion, a two-stage expansion was conducted with 5 L and 15 L stirred-tank bioreactors to yield 1.1 x 1010 hMSCs with an overall 128-fold expansion within 9 days. The cells were harvested by completely dissolving the microcarriers, concentrated, washed and formulated with a continuous-flow centrifuge-based cell processing system, and then aliquoted with a cell filling system. Compared with 2D planar culture, there are no significant differences in the quality of hMSCs harvested from 3D culture. We have also applied these dissolvable porous microcarriers to other popular cell types in the CGT sector; specifically, HEK293T cells and Vero cells have been cultivated to peak cell densities of 1.68 x 107 cells/mL and 1.08 x 107 cells/mL, respectively. This study provides a protocol for using a bioprocess engineering platform harnessing the characteristics of GMP-grade dissolvable microcarriers and advanced closed equipment to achieve the industrial-scale manufacturing of adherent cells.

Autor destacado: Avance en la fabricación de terapias celulares con microportadores solubles 

Here, we present a protocol to achieve the large-scale manufacturing of adherent cells through a fully closed system based on GMP-grade dissolvable microcarriers. The cultivation of human mesenchymal stem cells, HEK293T cells, and Vero cells was validated and met both quantity demands and quality criteria for the cell and gene therapy industry.

Producción celular a gran escala basada en microportadores porosos solubles de grado GMP

Researchers in the cell and gene therapy (CGT) industry have long faced a formidable challenge in the efficient and large-scale expansion of cells. To ...