Para Margaret CM Smith por gentilmente fornecer o clone integrase Apoio financeiro de: Genoma Canadá / Genoma Prairie NSERC Discovery e subsídios projeto estratégico

1. Produção de Cultura <ol><li> Preparar meios líquidos estéreis: TY 10 (5 g / l de triptona, extracto de levedura 3 g / l, 0,44 g / l de cloreto de cálcio hidratado), e LB 11 (10 g / l de triptona, 5 g de extracto de levedura / l, 5 g / cloreto de sódio, pH 7). </li><li> Inocular a partir de uma única colônia: SmUW227 (S. meliloti LP-deformação: a construção será descrito em outro lugar, a estirpe detalhes de construção disponíveis mediante solicitação) () em 5 ml de TY media com 50 mg / ml espectinomicina. Inocular as seguintes estirpes em 5 ml de meio líquido LB, que complementa com o antibiótico dado: E. coli DH5a 12 contendo pJC2, a expressão da integrase plasmídeo, com 10 ug / ml de tetraciclina; E. coli MT616 13, o mobilizador, com 10 ug / ml de cloranfenicol; e E. coli DH5a contendo pJH110, a cassete de plasmídeo dador, com 5 ug / ml de gentamicina. </li><li> Incubar E. cepas de Escherichia durante a noite a 37 e°; C com agitação constante. Incubar S. meliloti estirpe a 30 ° C durante dois dias, com agitação. É possível obter uma S. meliloti cultura durante a noite com uma maior inoculo (1:500 subcultura de uma cultura saturada). </li></ol> 2. Preparação Cultura e Mixing <ol><li> Lavar 1,5 ml de cultura para cada estirpe, recolhendo o sedimento de células por centrifugação a 17.000 xg durante 30 segundos e ressuspensão em 1 ml estéril NaCl 0,85%; repetição uma vez. </li><li> Ressuspender o sedimento em 100 uL de estéril NaCl 0,85%. </li><li> Individualmente manchar 10 ul de cultura lavada para cada estirpe em uma placa de agar TY liso. Estes pontos são pontos de controle. </li><li> Adicionar 40 ul de cada estirpe excluindo a E. coli DH5a 12 contendo pJC2 num tubo estéril, mistura, e uL local 120 desta mistura sobre uma placa de agar TY liso. Este ponto é o controle não integrase-negativo. </li><li> Adicionar 40 ul de cada estirpe em um t estérilube, misturar, e ponto 160 ul desta mistura em um prato simples de ágar TY. Este ponto é o local de acasalamento IMCE. </li><li> Permitir pontos para secar em uma capela de fluxo laminar. </li><li> Placas de vedação e incubar a 30 ° C durante a noite. </li></ol> 3. Isolamento de Trans-integrantes <ol><li> Manchas de registo contínuo de controlo e no local IMCE em placas de agar TY suplementado com 200 ug / ml de estreptomicina (SmUW227 baseia-se na Rm1021 que transporta uma mutação que confere alto nível de resistência à estreptomicina 14) e gentamicina 30 ug / ml. </li><li> Para o cálculo da eficiência IMCE, ressuspender aproximadamente ¼ do local de acasalamento IMCE em 500 ul de NaCl 0,85% estéril. Fazer 10 diluições em série de 10 -7. Diluições placa 10 por meio de 10 -5 -2 em placas de agar TY suplementado com 200 ug / ml de estreptomicina e 30 ug / ml de gentamicina, à selecção de trans-integrantes. Diluições placa 10 thr -3ough 10 -7 em placas de agar TY suplementado com 200 ug / ml de estreptomicina, para seleccionar para ambos os trans-integrantes e potenciais receptores, isto é, receptores totais. </li><li> Para o isolamento de markerless trans-integrantes ressuspender aproximadamente um quarto do local de acasalamento IMCE em 500 uL de estéril NaCl 0,85%. Fazer 10 diluições em série de 10 -6. Diluições placa 10 -4 a 10 -6 em quatro placas de agar TY suplementado com 200 ug / ml de estreptomicina e 200 ug / ml X-gluc. </li><li> Incubar as placas a 30 ° C durante 3 dias. </li><li> Visualização RFP trans-integrantes sob luz verde (525 nm) e através de um filtro vermelho (&gt; 610 nm) 15. </li><li> Calcula-se a eficiência IMCE por cento como CFU de trans-integrantes em comparação com o CFU de destinatários totais. Cerca de metade dos trans-integrantes terão sofrido troca cassete verdadeiro tornando-os brancos e espectinomicina sensível. </li><li> Para encontrar os markerless trans-integrantes (waqui o vector dador não contém Gm r como em pJH110) tela para uma colónia branca (um período de incubação prolongada, 1-2 dias adicionais à temperatura ambiente, irá aumentar a diferenciação de colónias por meio de X-gluc, isto é necessário em S. meliloti SmUW227), confirmar a sensibilidade da colónia espectinomicina por triagem de uma placa de agar TY sem antibióticos e uma placa de agar TY contendo 100 ug / ml espectinomicina. </li></ol> 4. Os resultados representativos Após três dias de incubação em TY suplementado com estreptomicina e gentamicina as estrias de controlo deve ter nenhum crescimento. A raia IMCE deve ter crescimento confluente na raia cabeça e muitas colónias sobre o segundo raia, como visto na Figura 2. A eficiência da integração-trans, expresso como a percentagem de trans-integrantes aos destinatários totais, deve estar no intervalo de 0,5%. Cerca de metade dos trans-integrantes será espectinomicina sensível e brancomostrando que eles foram submetidos a troca de cassetes verdade. Trans-integrantes contendo o testador cassete doador rfp de pJH110 deve exibir fluorescência RFP perceptível quando visto sob luz verde (525 nm) e através de um filtro vermelho (&gt; 610 nm) 15. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="A Figura 1"/> . Figura 1 ilustração mistura Conjugação: Aided pela expressão dos genes de transferência de pRK600, todos os plasmídeos são transferidos aleatoriamente a partir de célula para célula. Esta transferência resulta na criação de trans-integrantes na mistura, por meio de aquisição da estirpe LP-de os dois plasmídeos necessários para IMCE, a integrase (int) pJC2 plasmídeo ajudante eo pJH110 plasmídeo dador. A cassete de dador, do dador não-replicante plasmídeo (pJH110) é transferida através de ΦC31 actividade da integrase com os marcadores do LP-cassete no cromossoma, resultando na perda do LP-marcadores (SpR e uidA) e para a manutenção da cassete de dador (rfp e GM r) na resultante trans-integrante. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="A Figura 2"/> Figura 2. A: B placa de acoplamento mancha sobre uma placa de TY não selectivo mostrando misturas de células secas na superfície de agar:. Manchas listras de acoplamento na placa de estreptomicina-gentamicina-X-gluc, a partir de topo esquerdo no sentido horário nenhum controlo integrase, IMCE em S. meliloti, E. coli DH5a contendo controle pJC2, E. coli DH5a contendo pJH110 controlo, E. controle coli MT616, e S. . meliloti controle UW227 C: 10 diluição -2 de ressuspensão local de acasalamento em TY estreptomicina-X-agar glic D: 10. diluição -2 de ressuspensão local de acasalamento em ágar TY-estreptomicina gentamicina-X-glic (colônias azuis são recombinantes individuais, colônias brancas foram submetidos a verdadetroca cassete) E: 10. diluição -2 de ressuspensão acasalamento mancha na TY agar estreptomicina-X-gluc mostrando a falta de fluorescência F: 10. diluição -2 de ressuspensão local de acasalamento em agar TY-estreptomicina gentamicina-X-gluc mostrando dois níveis de fluorescência (colônias brilhantes correspondem a colônias azuis e têm maior expressão rfp presumivelmente devido ao promotor de leitura que a partir de seqüência do vetor, onde colônias ter sido submetido a verdadeiro troca-cassete conter RFP com apenas promotor sua imediata sem leitura por meio do promotor lac em o vector, que está ausente.).

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Não há conflitos de interesse declarados.

A técnica IMCE permite a eficiente integração de uma cassete de ADN único attB flanqueada no LP-locus de uma estirpe previamente manipulada. Uma vez que a construção desejada é clonado no lugar de rfp para criar a cassete de dador, a técnica não requer purificação de ADN e transformação subsequente, o que torna muito robusta. É crítico que controla o crescimento adequados estão incluídos, para ser determinada a resistência a antibióticos é devido à criação de trans-integrantes e ...

<table border="1"><tbody><tr><td> Nome do reagente </td><td> Companhia </td><td> Número de catálogo </td><td> Comentários </td></tr><tr><td> Estreptomicina </td><td> Bioshop Canada Inc. </td><td> STP101 </td><td></td></tr><tr><td> Espectinomicina </td><td> Bioshop Canada Inc. </td><td> <a href="https://ssl34.alentus.com/bioshopcanada/detail.asp?Pin=SPE201">SPE201</a> </td><td></td></tr><tr><td> Gentamicina </td><td> Bioshop Canada Inc. </td><td> GTA202 </td><td></td></tr><tr><td> Choramphenicol </td><td> Bioshop Canada Inc. </td><td> CLR201 </td><td></td></tr><tr><td> Tetraciclina </td><td> Bioshop Canada Inc. </td><td> TET701 </td><td></td></tr><tr><td> Canamicina </td><td> Bioshop Canada Inc. </td><td> KAN201 </td><td></td></tr><tr><td> Agar grau bacteriológica </td><td&gt; Bioshop Canada Inc. </td><td> AGR001 </td><td></td></tr><tr><td> Triptona </td><td> Bioshop Canada Inc. </td><td> TRP402 </td><td></td></tr><tr><td> Extracto de levedura </td><td> Bioshop Canada Inc. </td><td> YEX401 </td><td></td></tr><tr><td> Cloreto de Sódio </td><td> Bioshop Canada Inc. </td><td> SOD001 </td><td></td></tr><tr><td> Cloreto de cálcio </td><td> Bioshop Canada Inc. </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Ouro Biotecnologia Inc. </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli estirpe MT616 </td><td> Disponível em cima do pedido </td><td></td><td> Também é usado fora do nosso laboratório </td></tr><tr><td> E. coli estirpe pJC2 </td><td> Em casa, disponíveis mediante solicitação </td><td></td><td></td></tr><tr><td> E. coli estirpe pJH110 </td><td> Em casa, disponível por Request </td><td></td><td></td></tr><tr><td> SmUW227 tensão </td><td> Em casa, disponíveis mediante solicitação </td><td></td><td></td></tr></tbody></table>

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

O cromossoma bacteriano pode ser utilizado para manter de forma estável de ADN estranho na gama mega-base 1. Integração no cromossoma contorna questões tais como a replicação do plasmídeo, a estabilidade do plasmídeo, a incompatibilidade do plasmídeo, e variância número plasmídeo de cópia. Este método utiliza a integrase site-specific a partir do fago de Streptomyces (Φ) C31 2,3. A integrase ΦC31 catalisa uma recombinação direta entre dois sítios de DNA específicas: attB e attP (34 e 39 pb, respectivamente) 4. Esta recombinação é estável e não revertem 5. A &quot;pista&quot; sequência (LP), constituído por um gene de resistência à espectinomicina, aadA (SPR), ea E. gene coli ß-glucuronidase (uidA) ladeado por sites attP foi integrado nos cromossomos de Sinorhizobium meliloti, anthropi Ochrobactrum e Agrobacterium tumefaciens em uma região intergênica, a ampC locus, eo locus Teta, respectivamente. S. meliloti é usado neste protocolo. Vectores de dadores contendo locais attB mobilizável que flanqueiam um stuffer vermelho fluorescente proteína do gene (SDP) e um gene de resistência a antibiótico também têm sido construída. Neste exemplo, o pJH110 resistente gentamicina plasmídeo é usado. O gene rfp 6 pode ser substituída com uma construção desejada utilizando Sph I e Pst I. Alternativamente um construto sintético flanqueado por sítios attB podem ser sub-clonado num vector mobilizável, tais como pK19mob 7. A expressão do gene da integrase ΦC31 (clonado a partir de pHS62 8) é conduzida pelo promotor lac, em uma ampla gama de hospedeiros mobilizável plasmídeo pRK7813 9. Um protocolo de acasalamento tetraparental é usado para transferir a cassete de doador na estirpe LP substituindo assim os marcadores na sequência de LP com a cassete de dador. Estas células são trans-integrantes. Trans-integrantes são formados com uma eficiência típica de 0,5%. Trans-integrantes são tipicamente encontrados nos primeiros 500-1,000 colónias filtradas através da sensibilidade aos antibióticos ou azul-branco rastreio utilizando 5-bromo-4-cloro-3-indolil-beta-D-glucurónico ácido (X-gluc). Este protocolo contém os procedimentos de acasalamento e de seleção para criar e isolar trans-integrantes.

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

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

Um método rápido e eficiente para integrar DNA estranho de juros em cepas pré-fabricados receptoras, chamadas de tensões almofada de aterragem, é descrito. O método permite site-specific integração de uma cassete de ADN no locus engenharia aterragem almofada de uma estirpe dada, por meio de conjugação e expressão da integrase ΦC31.

Site-specific Engenharia cromossomo bacteriano: ΦC31 Integrase Mediated Cassette Exchange (IMCE)

The bacterial chromosome may be used to stably maintain foreign DNA in the mega-base range1. Integration into the chromosome circumvents issues such as ...

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

Research

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Bioengineering

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

15.5K visualizações.  University of Waterloo. Um método rápido e eficiente para integrar DNA estranho de juros em cepas pré-fabricados receptoras, chamadas de tensões almofada de aterragem, é descrito. O método permite site-specific integração de uma cassete de ADN no locus engenharia aterragem almofada de uma estirpe dada, por meio de conjugação e expressão da integrase ΦC31.

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

Procedure for Lung Engineering

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

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

Procedimento para a Engenharia de pulmão

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

Bioengenharia

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Caracterizando Voláteis bacteriana secundária usando ionização Espectrometria de Massa (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

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

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

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

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

Alta eficiência, Transfecção site-specific de células aderentes com siRNA Usando matrizes de microeletrodos (MEA)

The discovery of RNAi pathway in eukaryotes and the subsequent development of RNAi agents, such as siRNA and shRNA, have achieved a potent method for ...

Capillary Force Lithography for Cardiac Tissue Engineering

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

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

Capilar Força Litografia de Engenharia tecido cardíaco

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

Observing and Quantifying Fibroblast-mediated Fibrin Gel Compaction

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

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

Observando e quantificando compactação de gel de fibrina mediada por fibroblasto

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

Manufacturing Of Robust Natural Fiber Preforms Utilizing Bacterial Cellulose as Binder

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

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

Fabricação de fibras naturais robustos Preformas Utilizando bacteriana Celulose como Binder

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

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

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

Microbiologically Induced Calcite Precipitation Mediated by Sporosarcina pasteurii

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

Microbiologicamente induzida Calcita Precipitation Mediada por<em&gt; Sporosarcina pasteurii</em

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

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

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

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

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

Engenharia de Tecidos por Intrínseca Vascularização numa<em&gt; In Vivo</em&gt; Câmara Engenharia de Tecidos

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

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

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

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

Microfluídico troca de tampão para Micro / nanopartículas celular Engenharia sem interferências

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

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

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

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

Formação Light-mediada e padronização de hidrogéis para aplicações de cultura celular

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