Este trabalho foi financiado pela NSF RUI Grants MCB-0950372 e 1105310 DMR-intitulado &quot;Caracterização Molecular de Silks aranha viúva negra e comportamento mecânico de Spider Silks cola&quot;, respectivamente.

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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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As fibras sintéticas fiados a partir desta metodologia são mecanicamente sobre a mesma ordem de grandeza em comparação com as fibras naturais. Diminuindo a quantidade de erro humano por mecanização da spooling e processos pós tração de spin, a variação experimental entre as amostras são mais controlada e grandemente reduzida. A nossa metodologia oferece o potencial para investigar as propriedades mecânicas de outras fibras que são fiados a partir de proteínas recombinantes cod...

A seda da aranha tem extraordinárias propriedades mecânicas que se realiza vários materiais sintéticos, incluindo aço de alta resistência, Kevlar e Nylon. 1 Spiders girar pelo menos 6-7 diferentes tipos de fibras que exibem diversas propriedades mecânicas, cada um projetado com quantidades variadas de resistência à tração e extensibilidade para executar tarefas específicas biológicos. 2 cientistas da pesquisa são rapidamente buscar a utilização de sedas aranha como biomateriais próxima geração devido às suas excelentes propriedades mecânicas, a sua biocompatibilidade, ea sua natureza não-tóxico e verde-de material. 3,4 Devido à canibalista e natureza venenosa de aracnídeos, a colheita sedas de aranhas através da agricultura não é uma estratégia prática para atender as demandas necessárias para a fabricação em escala industrial. Portanto, os cientistas virou-se para a produção de proteínas recombinantes de seda em organismos transgénicos acoplado com in vitro fiação de fibras sintéticas a partir doproteínas purificadas. si só, 5-8 Expressão de full-length proteínas recombinantes de seda de aranha foi tecnicamente difícil, dadas as propriedades intrínsecas de suas seqüências de genes, que incluem sua natureza altamente repetitiva e comprimentos físicos (&gt; 15 kb), GC-rica de conteúdo e tendenciosa alanina e glicina utilização de codões. 9-11 Até à data, a maioria dos laboratórios têm-se centrado na expressão formas truncadas das proteínas principais ampullate seda MaSp1 ou MaSp2 utilizando sequências parciais de cDNA ou genes sintéticos. 12-15 Spinning sintético aranha sedas é um processo difícil que requer domínio e conhecimento em várias disciplinas científicas, e as complexidades do processo de fiação não foram totalmente revelados ao público em geral pela representação de vídeo. Na verdade, apenas um punhado de laboratórios em todo o mundo têm a expertise para expressar os cDNAs de seda de aranha, purificar as proteínas da seda, girar fibras sintéticas e fazer a pós spin-draw, e, finalmente, testar suas propriedades de biomateriais. 8,16,17 diferentes abordagens para fiação de fibras sintéticas abrangidas fiação úmida e seca, bem como métodos electrospinning 16,18,19 Todos os procedimentos têm um objetivo em comum -. Desenvolvimento de um protocolo que produz seda de aranha sintética com propriedades mecânicas que rivais tópicos naturais para grande escala processos de fabrico comercial. Aqui descrevemos o procedimento para gerar seda de aranha artificial em escala de laboratório usando uma metodologia de extrusão húmida. Em relação a outros métodos girando, girando molhadas tem produzido os resultados mais consistentes para a análise de fibra. Nós delinear este início procedimento com a expressão das proteínas recombinantes de seda em bactérias, seguido por sua purificação, e, em seguida, descrevem os passos de preparação de proteína para a fiação, incluindo uma metodologia draw pós-rotação aplicado à &quot;tal como fiados&quot; fibras que dá o tópicos com propriedades dos materiais que abordam a qualidade da seda de aranha naturais. Nosso METODOLÓGICASy é projetado para imitar o processo natural de fiação de fibras de seda e baseia a nossa experiência da arquitetura e da função das glândulas produtoras de seda do orbe e cob-aranhas tecendo 20-22. Além disso, podemos concluir com o necessário passos para determinar as propriedades do material das fibras sintéticas usando um tensometer para traçar curvas tensão-deformação, que permitem que os investigadores para calcular a resistência à tracção, a estirpe final, tenacidade e de fibras. Por último, mas de valor significativo, os aparelhos de fiação, o spooling e desenho podem ser construídos em casa usando as peças disponíveis no mercado, ao invés de comprar elaborado e caro equipamento personalizado.

<table border="1"><tbody><tr><td> Reagente / Equipamento </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> pBAD / TOPO Kit Expression ThioFusion </td><td> Invitrogen </td><td> K370-01 </td><td></td></tr><tr><td> Fastbreak reagente lise celular, 10x </td><td> Promega </td><td> V857C </td><td></td></tr><tr><td> Ni-NTA Agarose </td><td> Qiagen </td><td> 30210 </td><td> Inclui instruções para buffers </td></tr><tr><td> ProteoSilver Kit Prata Stain </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> Seringa </td&gt; <td> Hamilton </td><td> 7657-01 </td><td> 250 uL </td></tr><tr><td> Agulha </td><td> Hamilton </td><td> 7780-01 </td><td> Medidor de 26s, Blunt ponta de agulha removível </td></tr><tr><td> Bomba de seringa </td><td> Harvard Apparatus </td><td> 702208 </td><td> 11Plus </td></tr><tr><td> Digital Caliper </td><td> Carrera </td><td> CP5906 </td><td> 0-150 mm Gama </td></tr><tr><td> Fórceps em aço inoxidável </td><td> Instrumentos de precisão do mundo </td><td> 501764 </td><td> Mini Dumont # M5S </td></tr><tr><td> Motor </td><td> Moinho Natureza </td><td> 7090529 </td><td> 12VDC, 2 rpm </td></tr><tr><td> Atuador Linear </td><td> Warner Elétrica </td><td> 01-D024-0050-A06-LP-IP65 </td><td> 24VDC variando de 6 polegadas </td></tr><tr><td> Microscópio de dissecção </td><td> Leica Microsystems </td><td> Leica MZ16 </td><td></td></tr><tr><td> Digital Microsccâmera ope </td><td> Leica Microsystems </td><td> DFC320 </td><td> Software: Leica Application Suite v2.8.1 </td></tr><tr><td> Vannas tesoura </td><td> Instrumentos de precisão do mundo </td><td> 500260 </td><td></td></tr><tr><td> Microtensometer </td><td> Aurora Científico </td><td> 310C </td><td> 5N Sistema Dual-Mode </td></tr></tbody></table>

synthetic spider silk production on a laboratory scale

Como a sociedade progride e os recursos se tornam escassos, está se tornando cada vez mais importante para cultivar novas tecnologias que os biomateriais engenheiro de próxima geração com propriedades de alto desempenho. O desenvolvimento destes novos materiais estruturais deve ser rápida, custo-eficiente e envolvem metodologias de processamento e produtos que sejam ecologicamente corretos e sustentáveis. Aranhas giram uma multidão de diferentes tipos de fibra, com diversas propriedades mecânicas, oferecendo uma rica fonte de materiais de engenharia de última geração para biomimicry que rivalizam com os melhores materiais sintéticos e naturais. Como a coleta de grandes quantidades de seda de aranha natural é impraticável, a produção de seda sintética tem a capacidade de fornecer aos cientistas o acesso a um suprimento ilimitado de tópicos. Portanto, se o processo de fiação pode ser simplificado e aperfeiçoado, fibras artificiais de aranha têm o potencial de uso para uma ampla gama de aplicações que vão desde armaduras, sutura cirúrgicas, cordas e cabos, pneus, cordas para instrumentos musicais, e compósitos para a aviação e tecnologia aeroespacial. A fim de avançar o processo de produção de seda e sintéticos para produzir fibras que exibem variação baixa em suas propriedades materiais de rotação para girar, foi desenvolvido um protocolo de extrusão húmida que integra a expressão de proteínas recombinantes de seda de aranha em bactérias, purificação e concentração das proteínas , seguido por extrusão de fibras e um pós-tratamento de spin-mecânico. Esta é a primeira representação visual que revela um processo de passo-a-passo para girar e analisar fibras de seda artificial numa escala de laboratório. Ele também fornece detalhes para minimizar a introdução de variabilidade entre as fibras fiadas a partir da mesma droga de fiação. Coletivamente, estes métodos irá impulsionar o processo de produção de seda artificial, levando a fibras de alta qualidade que superam as sedas naturais de aranhas.

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

Apesar das propriedades mecânicas excelentes e bioquímicos da aranha sedas, este material não pode ser colhido em grandes quantidades por meios convencionais. Aqui nós descrevemos uma estratégia eficiente para produzir fibras de seda de aranha artificial, que é um processo importante para os investigadores que estudam a produção de seda de aranha e sua utilização como a próxima geração de biomateriais.

Produção de seda sintética Aranha em escala de laboratório

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 visualizações.  University of the Pacific. Apesar das propriedades mecânicas excelentes e bioquímicos da aranha sedas, este material não pode ser colhido em grandes quantidades por meios convencionais. Aqui nós descrevemos uma estratégia eficiente para produzir fibras de seda de aranha artificial, que é um processo importante para os investigadores que estudam a produção de seda de aranha e sua utilização como a próxima geração de biomateriais.

Vídeo: 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 ar de dispositivos, incluindo malhas não tecidos de electrospun Proteínas Recombinantes de seda de aranha

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

Bioengenharia

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.

Projetando proteína de seda-seda liga Materiais para aplicações 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.

Produção e Segmentação de 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.

Clonagem e produção em larga escala de alta capacidade vetores adenovirais com base no tipo adenovírus 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.

Baseada em Silk-colágeno Engineered 3D Model of Tissue polarizada Neural

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.

Entrada de poder de medições em biorreatores mexidas em escala de laboratório

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.

Formação de Material de Seda de Aranha Recombinante por Solvatação Aquosa Usando Calor e Pressão

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.

Produção automatizada em larga escala de esferoides de células-tronco derivados de adiposo para bioimpressão 3D

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

Produção em sistema fechado de células aderentes baseadas em microtransportadores solúveis

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 em destaque: Avançando na fabricação de terapia celular com microtransportadores solúveis 

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.

Produção de células em larga escala com base em microtransportadores porosos solúveis de grau 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 ...

Methods Article

Produção de seda sintética Aranha em escala de laboratório