Diese Arbeit wurde von der NSF RUI Grants MCB-0950372 und DMR-1105310 mit dem Titel &quot;Molekulare Charakterisierung von Black Widow Spider Silks und mechanische Verhalten von Spider Silks Kleber&quot;, bzw. unterstützt.

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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. 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Keine Interessenskonflikte erklärt.

Synthetische Fasern, die aus dieser Methode versponnen werden mechanisch auf der gleichen Größenordnung im Vergleich zu den natürlichen Fasern. Durch die Verringerung der Menge an menschlichem Versagen durch Mechanisierung der Spooling-und Post-Spin Auslosung Prozesse, sind die experimentelle Variation zwischen den Proben kontrollierter und stark reduziert. Unsere Methodik bietet die Möglichkeit, die mechanischen Eigenschaften der Fasern, die aus anderen rekombinanten Proteinen aus den c...

Spinnenseide verfügt über hervorragende mechanische Eigenschaften, die heraus führt mehrere künstlichen Materialien, einschließlich hochfestem Stahl, Kevlar und Nylon. 1 Spinnen spinnen mindestens 6-7 unterschiedliche Fasertypen, die unterschiedliche mechanische Eigenschaften zeigen, die jeweils mit unterschiedlichen Mengen an Zugfestigkeit und Erweiterbarkeit ausgelegt um spezifische biologische Aufgaben zu erfüllen. 2 Forschung Wissenschaftler werden schnell verfolgt die Verwendung von Spinnenseide als Biomaterial nächsten Generation aufgrund ihrer hervorragenden mechanischen Eigenschaften, ihrer Biokompatibilität und ihre nicht-toxisch und grün-materieller Natur. 3,4 Wegen des kannibalischen und giftigen Natur der Spinnentiere, Ernte Spinnenseide durch die Landwirtschaft ist keine praktische Strategie, um den Anforderungen, die für Herstellung im industriellen Maßstab gerecht zu werden. Daher haben Wissenschaftler in die Produktion von rekombinanten Seidenproteinen in transgenen Organismen in vitro gekoppelt Spinnen von synthetischen Fasern aus der eingeschaltetse gereinigte Proteine. 8.5 Expression von rekombinanten Volllängen-Spinnenseidenproteinen war technisch schwierig angesichts der Beschaffenheit ihrer Gensequenzen, die ihre stark repetitiven Natur und physikalischen Längen (&gt; 15 kb) umfassen, GC-reiche Inhalt und voreingenommen Alanin und Glycin Codonverwendung. 11.09 Bis heute haben die meisten Labors auf auszudrücken verkürzten Formen der großen Ampullendrüse Seidenproteine ​​MaSp1 oder MaSp2 mit partiellen cDNA-Sequenzen oder synthetische Gene konzentriert. 15.12 Spinning synthetischen Spinnenseide ist ein herausfordernder Prozess, der Beherrschung und Kenntnisse in verschiedenen wissenschaftlichen Disziplinen und den Feinheiten der Spinnverfahren sind noch nicht vollständig an die breite Öffentlichkeit von Video-Darstellung offenbart. In der Tat haben nur eine Handvoll Labors auf der ganzen Welt das Know-how, um die Spinnenseide cDNAs zum Ausdruck bringen, reinigen die Seidenproteine, synthetischen Fasern zu spinnen, und führen Sie nach dem Spin-Unentschieden, und dann schließlich testen ihre Eigenschaften Biomaterial. 8,16,17 Unterschiedliche Ansätze für die Spinnerei synthetischen Fasern haben umfasste nassen und trockenen Spinnerei sowie Elektrospinnen Methoden 16,18,19 Alle Verfahren haben ein gemeinsames Ziel -. Entwicklung eines Protokolls, das synthetische Spinnenseide produziert mit mechanischen Eigenschaften, die rivalisierenden natürlichen Fäden für groß angelegte kommerzielle Herstellungsverfahren. Hier beschreiben wir den Vorgang, um künstliche Spinnenseide im Labormaßstab mit Hilfe eines Nassverspinnen Methodik zu generieren. Im Vergleich zu anderen Spinnverfahren hat Naßspinnen die konstantesten Ergebnisse für Faser-Analyse erzeugt. Wir stellen diese Vorgehensweise, beginnend mit der Expression der rekombinanten Proteine ​​in Bakterien Seide, die durch ihre anschließende Reinigung und beschreiben anschließend die Schritte zur Vorbereitung Protein für die Spinnerei, einschließlich einer post-Spin-Methode wie für die Auslosung &quot;, wie gesponnen&quot; Fasern, Fäden ergibt sich mit Materialeigenschaften, die die Qualität der natürlichen Spinnenseide zu nähern. Unsere methodology wurde entwickelt, um genau imitieren den natürlichen Spinnprozess von Seidenfasern und es greift stark auf unser Know-how über die Architektur und Funktion der Seide-produzierende Drüsen von Orb-und COB-Weben Spinnen. 20-22 Darüber hinaus schließen wir mit der notwendigen Schritte, um die Materialeigenschaften der synthetischen Fasern mit einem Dehnungsmesser zu Spannungs-Dehnungs-Kurven, die Ermittler auf die Zugfestigkeit, Bruchdehnung und Zähigkeit der Fasern berechnen lassen plotten zu bestimmen. Schließlich aber von bedeutendem Wert, können die Spinnen, Spulen, Apparate und Zeichnung sein Zuhause gebaut mit handelsüblichen Teilen erwarben, anstatt aufwendige und teure kundenspezifische Geräte.

<table border="1"><tbody><tr><td> Reagenz / Ausrüstung </td><td> Firma </td><td> Katalog-Nummer </td><td> Kommentare </td></tr><tr><td> pBAD / TOPO Expression Kit ThioFusion </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> FastBreak Cell Lysis Reagent, 10x </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> Ni-NTA-Agarose </td><td> Qiagen </td><td> 30210 </td><td> Inklusive Anleitung für Puffer </td></tr><tr><td> ProteoSilver Silver Stain Kit </td><td> Sigma-Aldrich </td><td> PROTSIL1-1KT </td><td></td></tr><tr><td> FreeZone Lyophilisierer </td><td> Labconco </td><td> 7960041 </td><td> FreeZone 12plus </td></tr><tr><td> Hexafluorisopropanol (HFIP) </td><td> Sigma-Aldrich </td><td> 52512 </td><td></td></tr><tr><td> Spritze </td&gt; <td> Hamilton </td><td> 7657-01 </td><td> 250 ul </td></tr><tr><td> Nadel </td><td> Hamilton </td><td> 7780-01 </td><td> 26s Gauge, Blunt Ende auswechselbarer Nadel </td></tr><tr><td> Spritzenpumpe </td><td> Harvard Apparatus </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> Digitaler Messschieber </td><td> Carrera </td><td> CP5906 </td><td> 0-150 mm-Bereich </td></tr><tr><td> Edelstahlpinzette </td><td> World Precision Instruments </td><td> 501764 </td><td> Mini Dumont # M5S </td></tr><tr><td> Motor </td><td> Natur Mill </td><td> 7090529 </td><td> 12VDC, 2 min-Geschwindigkeit </td></tr><tr><td> Linear Actuator </td><td> Warner Electric </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC, 6-Zoll-Bereich </td></tr><tr><td> Seziermikroskop </td><td> Leica Microsystems </td><td> Leica MZ16 </td><td></td></tr><tr><td> Digitales MikroskopOPE-Kamera </td><td> Leica Microsystems </td><td> DFC320 </td><td> Software: Leica Application Suite v2.8.1 </td></tr><tr><td> Vannas Schere </td><td> World Precision Instruments </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> Aurora Scientific </td><td> 310C </td><td> 5N Dual-Mode-System </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

Wie die Gesellschaft fortschreitet und die Ressourcen werden knapper, wird es immer wichtiger, neue Technologien zu kultivieren, dass Ingenieur Biomaterialien nächsten Generation mit hoher Performance Eigenschaften. Die Entwicklung dieser neuen strukturellen Materialien muss schnell, kostengünstig und beinhalten Verarbeitungsverfahren und Produkte, die umweltfreundlich und nachhaltig sind. Spinnen spinnen eine Vielzahl von verschiedenen Fasertypen mit unterschiedlichen mechanischen Eigenschaften und bietet eine reiche Quelle der nächsten Generation technischen Werkstoffen für Bionik, dass Rivalen die besten künstlichen und natürlichen Materialien. Da die Aufnahme von großen Mengen der natürlichen Spinnenseide ist unpraktisch, hat Kunstseide Produktion die Möglichkeit, Wissenschaftler mit Zugang zu einem unbegrenzten Vorrat an Themen zu bieten. Deshalb, wenn das Spinnverfahren können gestrafft und perfektioniert haben künstliche Spinne Fasern die mögliche Verwendung für ein breites Spektrum von Anwendungen, die von Body Armor, chirurgisches Nahtmaterials, Kabel und Seile, Reifen, Saiten für Musikinstrumente, und Verbundwerkstoffe für Luft-und Raumfahrttechnik. Um die Kunstseide Produktion voranzubringen und zu Fasern, die geringe Varianz zeigen in ihren materiellen Eigenschaften von Spin zu drehen nachgeben, entwickelten wir eine Nassverspinnen Protokoll, das die Expression von rekombinanten Spinnenseide Proteine ​​in Bakterien, Reinigung und Konzentration der Proteine ​​integriert , gefolgt von Faserextrusion und mechanische Nachbearbeitung Spin Behandlung. Dies ist das erste visuelle Darstellung, die eine Schritt-für-Schritt-Prozess zeigt sich zu drehen und zu analysieren, Kunstseide Fasern im Labormaßstab. Es liefert auch Informationen, um die Einführung der Variabilität zwischen den Fasern aus dem gleichen Spinnlösung versponnen zu minimieren. Zusammen werden diese Methoden treiben den Prozess der Kunstseide Produktion, was zu höherer Qualität Fasern, die natürliche Spinnenseide zu übertreffen.

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

Trotz der hervorragenden mechanischen und biochemischen Eigenschaften von Spinnenseide, kann dieses Material nicht in großen Mengen mit konventionellen Mitteln gewonnen werden. Hier beschreiben wir eine wirksame Strategie, um künstliche Spinnenseide, die ein wichtiger Prozess für die Ermittler Studium Spinnenseide Herstellung und ihre Verwendung als Next-Generation-Biomaterialien ist zu spinnen.

Synthetische Spinnenseide Produktion im Labormaßstab

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 Aufrufe.  University of the Pacific. Trotz der hervorragenden mechanischen und biochemischen Eigenschaften von Spinnenseide, kann dieses Material nicht in großen Mengen mit konventionellen Mitteln gewonnen werden. Hier beschreiben wir eine wirksame Strategie, um künstliche Spinnenseide, die ein wichtiger Prozess für die Ermittler Studium Spinnenseide Herstellung und ihre Verwendung als Next-Generation-Biomaterialien ist zu spinnen.

Serie: 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.

Luftfilter Devices Inklusive Vlies Meshes of Elektrogesponnene Rekombinante Spinnenseidenproteine

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

Forschung

Biotechnik

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.

Gestaltung Silk-Seidenprotein-Legierungen für biomedizinische Anwendungen

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.

Produktion und Targeting von Monovalenter Quantenpunkte

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.

Klonierung und großtechnische Produktion von High-Capacity Adenovirusvektoren Auf der Grundlage des humanen Adenovirus Typ 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.

Engineered 3D Silk-Kollagen-basierten Modell von polarisiertem 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 ...

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.

Eingabe Leistungsmessungen in gerührten Bioreaktoren im Labormaßstab

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.

Materialbildung von Rekombinanten Spider-Seiden durch wässrige Lösung mit Wärme und Druck

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.

Großflächige, automatisierte Produktion von aus Fett gewonnenen Stammzellsphäroiden für den 3D-Bioprinting

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

Geschlossene Systemproduktion von adhärenten Zellen auf Basis von löslichen Microcarriern

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 im Rampenlicht: Weiterentwicklung der Herstellung von Zelltherapien mit auflösbaren Microcarriern 

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.

Großtechnische Zellproduktion auf Basis von auflösbaren porösen Mikroträgern in GMP-Qualität

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 ...