Ce travail a été soutenu par la NSF Subventions RUI MCB-0950372 et 1105310 DMR-droit &quot;Caractérisation moléculaire de Black Widow Soies d&#39;araignée et le comportement mécanique des Soies d&#39;araignée colle», respectivement.

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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>

Pas de conflits d&#39;intérêt déclarés.

Les fibres synthétiques filées à partir de cette méthodologie sont mécaniquement sur le même ordre de grandeur par rapport aux fibres naturelles. En diminuant la quantité d&#39;erreur humaine en mécanisant le bobinage et les processus post-tirage de spin, la variation expérimentale entre les échantillons sont plus contrôlée et fortement réduit. Notre méthodologie offre la possibilité d&#39;enquêter sur les propriétés mécaniques des fibres d&#39;autres qui sont filés à pa...

Soie de l&#39;araignée a des propriétés mécaniques extraordinaires qui effectue des matériaux synthétiques, y compris plusieurs en acier haute résistance, le kevlar et le nylon. 1 araignées tissent au moins 6-7 différents types de fibres qui affichent diverses propriétés mécaniques, chacun étant conçu avec des quantités variables de résistance à la traction et l&#39;extensibilité pour effectuer des tâches spécifiques. biologiques 2 Les chercheurs sont rapidement la poursuite de l&#39;utilisation de la soie d&#39;araignée en tant que biomatériaux de la prochaine génération en raison de leurs excellentes propriétés mécaniques, leur biocompatibilité et leur nature non-toxiques et vert-matériau. 3,4 En raison de la cannibale et la nature venimeuse des arachnides, la récolte des soies d&#39;araignée à travers l&#39;agriculture n&#39;est pas une stratégie concrète pour répondre aux exigences nécessaires à la fabrication à l&#39;échelle industrielle. Par conséquent, les scientifiques se sont tournés vers la production de protéines recombinantes de soie dans des organismes transgéniques couplé avec in vitro la filature de fibres synthétiques à partir de lasoi protéines purifiées. 5-8 Expression de pleine longueur des protéines recombinantes de soie d&#39;araignée a été techniquement difficile compte tenu des propriétés intrinsèques de leurs séquences de gènes, qui comprennent leur nature hautement répétitive et les longueurs physiques (&gt; 15 kb), riche en GC contenu et partiale alanine et la glycine codon usage 9-11. À ce jour, la plupart des laboratoires ont mis l&#39;accent sur ​​l&#39;expression de formes tronquées des protéines majeures de soie ampullaires MASP1 ou MaSp2 utilisant des séquences partielles d&#39;ADNc ou des gènes synthétiques. 12-15 Spinning synthétique araignée soie est un processus difficile qui exige maîtrise et des connaissances dans plusieurs disciplines scientifiques, et les subtilités du processus de filage n&#39;ont pas été pleinement révélé au grand public par la représentation vidéo. En fait, seule une poignée de laboratoires à travers le monde ont l&#39;expertise nécessaire pour exprimer les ADNc de soie d&#39;araignée, de purifier les protéines de soie, le filage de fibres synthétiques et des fonctions de post-spin tirage, et puis finalement de tester leurs propriétés de biomatériaux. 8,16,17 Différentes approches pour le filage de fibres synthétiques ont englobé filage au mouillé et sec ainsi que des méthodes électrofilage 16,18,19 Toutes les procédures ont un but commun -. Élaboration d&#39;un protocole qui produit la soie d&#39;araignée synthétique ayant des propriétés mécaniques qui rivalisent avec les fils naturels à grande échelle des processus de fabrication commerciale. Nous décrivons ici la procédure pour générer des soies d&#39;araignée artificielle sur une échelle de laboratoire en utilisant une méthodologie de filage au mouillé. Par rapport aux autres procédés de filage, filage au mouillé a produit les résultats les plus cohérents pour l&#39;analyse des fibres. On exposer cette procédure commençant avec l&#39;expression des protéines de soie recombinant dans des bactéries, suivie par leur purification, puis décrire les étapes de préparation de protéines pour la filature, comprenant une méthodologie de post-étirage de spin appliquée à &quot;comme&quot; fibres filées et qui donne fils avec propriétés des matériaux qui se rapprochent de la qualité de la soie d&#39;araignée naturelle. Notre démarche méthodologiquey est conçu pour imiter au mieux le processus naturel de filage de fibres de soie et il s&#39;appuie largement sur ​​notre expertise de l&#39;architecture et la fonction des glandes produisant la soie de l&#39;ORB-et torchis tissage araignées. 20-22 De plus, nous concluons avec la nécessaire des mesures pour déterminer les propriétés des matériaux des fibres synthétiques en utilisant un tensiomètre pour tracer courbes contrainte-déformation, qui permettent aux enquêteurs de calculer la résistance à la rupture, déformation ultime, et la ténacité des fibres. Enfin, mais de grande valeur, les appareils de filature, bobinage, et le dessin peuvent être à la maison construit en utilisant des pièces disponibles dans le commerce, plutôt que l&#39;achat de matériel élaboré et coûteux personnalisé.

<table border="1"><tbody><tr><td> Réactif / Equipement </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> pBAD / TOPO Kit Expression ThioFusion </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> Réactif de lyse cellulaire FastBreak, 10x </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> Ni-NTA agarose </td><td> Qiagen </td><td> 30210 </td><td> Comprend les instructions pour tampons </td></tr><tr><td> ProteoSilver Argent Stain Kit </td><td> Sigma-Aldrich </td><td> PROTSIL1-1kt </td><td></td></tr><tr><td> FreeZone Lyophiliseur </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> Seringue </td&gt; <td> Hamilton </td><td> 7657-01 </td><td> 250 uL </td></tr><tr><td> Aiguille </td><td> Hamilton </td><td> 7780-01 </td><td> Gauge 26 ans, Blunt extrémité amovible aiguille </td></tr><tr><td> Pousse-seringue </td><td> Harvard Apparatus </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> Pied à coulisse digital </td><td> Carrera </td><td> CP5906 </td><td> 0-150 mm large </td></tr><tr><td> En acier inoxydable pince </td><td> Instruments de précision du monde </td><td> 501764 </td><td> Mini Dumont # M5S </td></tr><tr><td> Moteur </td><td> Mill Nature </td><td> 7090529 </td><td> 12VDC, 2 vitesses rpm </td></tr><tr><td> Actionneur linéaire </td><td> Warner Electric </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC, plage de 6 pouces </td></tr><tr><td> Microscope à dissection </td><td> Leica Microsystems </td><td> Leica MZ16 </td><td></td></tr><tr><td> Numérique Microsccaméra OPE </td><td> Leica Microsystems </td><td> DFC320 </td><td> Logiciel: Leica Application Suite v2.8.1 </td></tr><tr><td> Ciseaux Vannas </td><td> Instruments de précision du monde </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> Aurora scientifique </td><td> 310C </td><td> 5N Dual-Mode Système </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

Comme la société progresse et les ressources se raréfient, il est de plus en plus important de cultiver de nouvelles technologies qui biomatériaux ingénieur de la prochaine génération avec des propriétés de haute performance. Le développement de ces nouveaux matériaux structurels doit être rapide, rentable et sur les méthodologies de traitement et des produits qui sont respectueux de l&#39;environnement et durable. Araignées tissent une multitude de différents types de fibres avec diverses propriétés mécaniques, offrant une riche source de matériaux d&#39;ingénierie prochaines génération pour le biomimétisme qui rivalisent avec les meilleurs matériaux naturels et anthropiques. Depuis la collecte de grandes quantités de soie d&#39;araignée naturelle n&#39;est pas possible, la production de soie synthétique a la capacité de fournir aux scientifiques d&#39;avoir accès à un approvisionnement illimité de threads. Par conséquent, si le procédé de filage peut être simplifié et perfectionné, les fibres d&#39;araignée artificielle ont le potentiel de l&#39;utilisation pour un large éventail d&#39;applications allant des gilets pare-balles, suture chirurgicales, cordes et cordages, les pneus, les cordes pour instruments de musique, et les matériaux composites pour l&#39;aviation et de la technologie aérospatiale. Afin de faire progresser le processus de soie synthétique de production et de céder des fibres qui affichent une faible variance dans leurs propriétés des matériaux à partir de spin à tourner, nous avons développé un protocole de filage au mouillé qui intègre l&#39;expression de protéines recombinantes de soie d&#39;araignée dans les bactéries, la purification et la concentration des protéines , suivie par extrusion de fibres et une post-rotation mécanique de traitement. Il s&#39;agit de la première représentation visuelle qui révèle un processus étape par étape pour faire tourner et d&#39;analyser les fibres de soie artificielle sur une échelle de laboratoire. Il fournit également des détails afin de minimiser l&#39;introduction de la variabilité entre les fibres filées à partir de la dope même filature. Collectivement, ces méthodes va propulser le processus de production de la soie artificielle, ce qui conduit à des fibres de meilleure qualité qui dépassent les soies d&#39;araignée naturelle.

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

Malgré les excellentes propriétés mécaniques et biochimiques de soies de l&#39;araignée, ce matériau ne peut être récolté en grandes quantités par des moyens conventionnels. Nous décrivons ici une stratégie efficace pour faire tourner fibres artificielles de soie d&#39;araignée, ce qui est un processus important pour les enquêteurs qui étudient la production de soie d&#39;araignée et de leur utilisation en tant que prochaine génération de biomatériaux.

Synthétique de la production soie d&#39;araignée sur l&#39;échelle du laboratoire

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 vues.  University of the Pacific. Malgré les excellentes propriétés mécaniques et biochimiques de soies de l'araignée, ce matériau ne peut être récolté en grandes quantités par des moyens conventionnels. Nous décrivons ici une stratégie efficace pour faire tourner fibres artificielles de soie d'araignée, ce qui est un processus important pour les enquêteurs qui étudient la production de soie d'araignée et de leur utilisation en tant que prochaine génération de biomatériaux.

Vidéo: 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.

Filtre à air dispositifs, y compris maillages non tissé de électrofilées protéines recombinantes de soie d&#39;araignée

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

Recherche

Bio-ingénierie

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.

Concevoir protéines de soie soie Matériaux alliage pour applications biomédicales

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.

La production et le ciblage des monovalent 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.

Clonage et Production à grande échelle de haute capacité vecteurs adénoviraux Basé sur le type adénovirus humain 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.

Modèle de polarisée Neural Tissue base-Silk-collagène ingénierie 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.

Mesures d’entrée de puissance dans des bioréacteurs brassées à l’échelle du laboratoire

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.

Formation matérielle de soies d’araignée recombinantes par solvatation aqueuse utilisant la chaleur et la pression

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.

Production automatisée à grande échelle de sphéroïdes de cellules souches dérivées de cellules adipeuses pour la bioimpression 3D

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

Production en système fermé de cellules adhérentes à base de microtransporteurs 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.

Pleins feux sur l’auteur : Faire progresser la fabrication de thérapies cellulaires avec des microtransporteurs 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.

Production de cellules à grande échelle à base de microporteurs poreux solubles de qualité 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 ...