Pour Margaret Smith CM d&#39;avoir bien voulu fournir le clone de l&#39;intégrase Le soutien financier de: Génome Canada / Génome Prairie La découverte du CRSNG et des subventions de projets stratégiques

1. La production de la culture <ol><li> Préparer stériles milieux liquides: TY 10 (5 g / l de tryptone, extrait de levure 3 g / l, 0,44 g / l de chlorure de calcium dihydraté), et LB 11 (10 g / l de tryptone, extrait de levure 5 g / l à 5 g / le chlorure de sodium, pH 7). </li><li> Inoculer à partir d&#39;une seule colonie: SmUW227 (S. meliloti LP-souche: la construction sera décrite ailleurs, la souche de détails de construction sont disponibles sur demande) () dans 5 ml de TY médias avec 50 pg / ml de spectinomycine. Ensemencer les souches suivantes dans 5 ml de milieu liquide LB, additionnel avec l&#39;antibiotique donné: E. coli DH5a 12 contenant pJC2, l&#39;expression de l&#39;intégrase plasmide, avec 10 pg / ml de tetracycline; E. coli MT616 13, le mobilisateur, avec 10 pg / ml chloramphénicol, et E. coli DH5a contenant pJH110, la cassette plasmide donneur, avec 5 pg / ml de gentamicine. </li><li> Incuber E. Les souches de E. nuit à 37 &amp;deg; C avec une agitation constante. Incuber S. souche meliloti à 30 ° C pendant deux jours avec agitation. Il est possible d&#39;obtenir un S. la culture meliloti nuit avec un plus grand inoculum (1:500 sous-culture d&#39;une culture saturée). </li></ol> 2. Préparation de la culture et de mixage <ol><li> Laver 1,5 ml de culture pour chaque souche par la collecte du culot cellulaire par centrifugation à 17 000 xg pendant 30 secondes et la remise en suspension dans 1 ml stérile 0,85% de NaCl; répétition une fois. </li><li> Resuspendre le culot dans 100 ul de solution stérile de NaCl à 0,85%. </li><li> Individuellement repérer 10 ul de la culture lavée pour chaque souche sur une plaque de gélose TY plaine. Ces taches sont des taches de contrôle. </li><li> Ajouter 40 ul de chaque souche à l&#39;exclusion du E. coli DH5a 12 contenant pJC2 dans un tube stérile, mélange, et la tache 120 pi de ce mélange sur une plaque de gélose TY plaine. Cet endroit est le contrôle non-négatif de l&#39;intégrase. </li><li> Ajouter 40 ul de chaque souche dans un t stérileube, mélange, et la tache 160 ul de ce mélange sur une plaque de gélose TY plaine. Cet endroit est le lieu d&#39;accouplement IMCE. </li><li> Permettez-taches à sécher dans un hotte à flux laminaire. </li><li> Plaques d&#39;étanchéité et incuber à 30 ° C pendant la nuit. </li></ol> 3. Isolement de Trans-intégrants <ol><li> Taches de contrôle Streak et place IMCE sur des plaques d&#39;agar TY complété avec 200 ug / ml de streptomycine (SmUW227 est basé sur Rm1021 qui porte une mutation conférant une résistance de haut niveau à la streptomycine 14) et 30 pg / ml de gentamicine. </li><li> Pour le calcul de l&#39;efficacité IMCE, resuspendre environ ¼ de la tache d&#39;accouplement IMCE dans 500 ul stérile à 0,85% de NaCl. Assurez-10-dilutions à 10 -7. Dilutions 10 -2 plaques par 10 -5 sur gélose TY complétées avec 200 ug / ml de streptomycine et 30 ug / ml de gentamicine, de sélectionner pour trans-intégrants. Dilutions plaques 10 -3 through 10 -7 sur gélose TY complétées avec 200 ug / ml de streptomycine, de sélectionner pour les deux trans-intégrants et les bénéficiaires potentiels, à savoir les bénéficiaires au total. </li><li> Pour l&#39;isolement de sans marqueurs trans-intégrants resuspendre environ un quart de la tache d&#39;accouplement IMCE dans 500 pi de solution stérile de NaCl à 0,85%. Assurez-10-dilutions à 10 -6. Dilutions plaques 10 -4 à 10 -6 sur quatre des plaques d&#39;agar TY complétées avec 200 ug / ml de streptomycine et 200 pg / ml X-gluc. </li><li> Incuber les plaques à 30 ° C pendant 3 jours. </li><li> Voir DP trans-intégrants sous la lumière verte (525 nm) et à travers un filtre rouge (&gt; 610 nm) 15. </li><li> Calculer l&#39;efficacité IMCE pour cent en UFC de trans-intégrants par rapport à la UFC de bénéficiaires au total. Environ la moitié des trans-intégrants aura subi un échange de cassette vrai ce qui les rend blanche et à la spectinomycine sensible. </li><li> Pour trouver les sans marqueurs trans-intégrants (wici le vecteur donneur ne contient pas de Gm r comme dans pJH110) écran pour une colonie blanche (une période d&#39;incubation prolongée, 1-2 jours supplémentaires à la température ambiante, permettra d&#39;améliorer la différenciation des colonies par X-gluc, cela est nécessaire dans S. meliloti SmUW227), confirmer la sensibilité à la spectinomycine de la colonie par le dépistage sur une plaque de gélose TY sans antibiotiques et une plaque de gélose TY contenant 100 ug / ml de spectinomycine. </li></ol> 4. Les résultats représentatifs Après trois jours d&#39;incubation sur TY complétée avec la streptomycine et la gentamicine les stries de contrôle ne devrait pas avoir de croissance. La série IMCE devrait avoir une croissance confluente sur la strie la tête et de nombreuses colonies sur la strie seconde, comme on le voit dans la figure 2. L&#39;efficacité de la trans-intégration, exprimée en pourcentage de la trans-intégrants pour les bénéficiaires au total, devrait être de l&#39;ordre de 0,5%. Environ la moitié des trans-intégrants sera spectinomycine sensibles et blancattestant qu&#39;ils ont subi un échange de cassette vrai. Trans-intégrants contenant la cassette testeur donateurs rfp de pJH110 devrait afficher discernable fluorescence DP lorsqu&#39;on les examine sous la lumière verte (525 nm) et à travers un filtre rouge (&gt; 610 nm) 15. <img src="/files/ftp_upload/3698/3698fig1.jpg" alt="Figure 1"/> . Figure 1 illustre un mélange Conjugaison: Aidé par l&#39;expression des gènes de transfert de pRK600, tous les plasmides sont transférés au hasard de la cellule à cellule. Ce transfert se traduit par la création de trans-intégrants dans le mélange, grâce à l&#39;acquisition de la LP-souche de ces deux plasmides requis pour IMCE, l&#39;intégrase (int) pJC2 plasmide auxiliaire et le pJH110 plasmide donneur. La cassette des bailleurs de fonds du donneur non-réplication plasmidique (pJH110) sont échangées via ΦC31 activité de l&#39;intégrase avec les marqueurs de la LP-cassette sur le chromosome, ce qui entraîne la perte de la LP-marqueurs (Spr et uidA) et le maintien de la cassette des bailleurs de fonds (DP et Gm r) dans le trans-résultante intégrante. <img src="/files/ftp_upload/3698/3698fig2.jpg" alt="Figure 2"/> Figure 2. A: B plaque à godets accouplement sur ​​une plaque non-sélective TY montrant mélanges de cellules séchées à la surface de la gélose:. Points d&#39;accouplement striées de la streptomycine-gentamicine-X-gluc plaque, en haut à gauche dans le sens horaire à partir aucun contrôle de l&#39;intégrase, IMCE en S. meliloti, E. coli DH5a contenant le contrôle pJC2, E. coli contenant DH5a pJH110 commande, E. coli MT616 de contrôle, et S. . meliloti UW227 de contrôle C: 10 -2 dilution de la remise en suspension place d&#39;accouplement sur ​​TY streptomycine-X-gluc agar D:. 10 -2 dilution de la remise en suspension place d&#39;accouplement sur ​​les TY-streptomycine gentamicine-X-gluc agar (colonies bleues sont recombinants simples, colonies blanches ont fait l&#39;objet vrai. échange cassette) E: 10 -2 dilution de la remise en suspension place d&#39;accouplement sur ​​TY streptomycine-X-gluc agar montrant l&#39;absence de fluorescence F:. 10 -2 dilution de la remise en suspension place d&#39;accouplement sur ​​les TY-streptomycine gentamicine-X-gluc agar montrant deux niveaux de fluorescence (brillantes colonies correspondent à des colonies bleues et ont une expression plus élevée rfp probablement à cause de promoteur lecture de la séquence si vecteur, où les colonies ayant subi vrai-cassette d&#39;échange contiennent des DP avec seulement son promoteur immédiate sans lecture par le biais du promoteur lac dans le vecteur, qui est absent.).

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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+insertion+and+replacement+in+Schizosaccharomyces+pombe+mediated+by+the+Streptomyces+bacteriophage+FC31+site-specific+recombination+system.">Gene insertion and replacement in Schizosaccharomyces pombe mediated by the Streptomyces bacteriophage FC31 site-specific recombination system.</a> Molecular Genetics and Genomics. 265, 1031-1038 (2001).</li><li>Katzen, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gateway+recombinational+cloning:+a+biological+operating+system.">Gateway recombinational cloning: a biological operating system.</a> Expert Opin. Drug Discovery. , 571-586 (2007).</li><li>Charles, T. C., Doty, S. L., Nester, E. 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Pas de conflits d&#39;intérêt déclarés.

La technique IMCE permet l&#39;intégration efficace d&#39;une cassette d&#39;ADN flanqué attB unique dans le LP-lieu d&#39;une souche auparavant conçu. Une fois la construction désirée est cloné en place de la DP pour créer la cassette des bailleurs de fonds, la technique ne nécessite pas de purification ultérieure de l&#39;ADN et de la transformation, ce qui rend très robuste. Il est essentiel que les contrôles de croissance appropriés sont inclus, pour être certain de la résistance aux ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Entreprise </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Streptomycine </td><td> Bioshop Canada Inc </td><td> STP101 </td><td></td></tr><tr><td> Spectinomycine </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> La gentamicine </td><td> Bioshop Canada Inc </td><td> GTA202 </td><td></td></tr><tr><td> Choramphénicol </td><td> Bioshop Canada Inc </td><td> CLR201 </td><td></td></tr><tr><td> Tétracycline </td><td> Bioshop Canada Inc </td><td> TET701 </td><td></td></tr><tr><td> Kanamycine </td><td> Bioshop Canada Inc </td><td> KAN201 </td><td></td></tr><tr><td> Bactériologiques agar de qualité </td><td&gt; Bioshop Canada Inc </td><td> AGR001 </td><td></td></tr><tr><td> Tryptone </td><td> Bioshop Canada Inc </td><td> TRP402 </td><td></td></tr><tr><td> Extrait de levure </td><td> Bioshop Canada Inc </td><td> YEX401 </td><td></td></tr><tr><td> Chlorure de sodium </td><td> Bioshop Canada Inc </td><td> SOD001 </td><td></td></tr><tr><td> Chlorure de calcium </td><td> Bioshop Canada Inc </td><td> CCL444 </td><td></td></tr><tr><td> X-gluc </td><td> Or Biotechnology Inc </td><td> G1281C1 </td><td></td></tr><tr><td> E. coli MT616 souche </td><td> Disponible sur demande </td><td></td><td> Également utilisé en dehors de notre laboratoire </td></tr><tr><td> E. coli souche pJC2 </td><td> Dans la maison, disponibles sur demande </td><td></td><td></td></tr><tr><td> E. coli souche pJH110 </td><td> Dans la maison, disponible en request </td><td></td><td></td></tr><tr><td> SmUW227 souche </td><td> Dans la maison, disponibles sur demande </td><td></td><td></td></tr></tbody></table>

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

Le chromosome bactérien peut être utilisé pour maintenir de façon stable l&#39;ADN étranger dans la plage méga-base 1. Intégration dans le chromosome contourne questions comme la réplication du plasmide, le plasmide de la stabilité, incompatibilité plasmidique, et le plasmide de la variance du nombre de copies. Cette méthode utilise l&#39;intégrase spécifique de site à partir du phage Streptomyces (Φ) C31 2,3. L&#39;intégrase catalyse une recombinaison ΦC31 direct entre deux sites d&#39;ADN spécifiques: attB et attP (34 et 39 pb, respectivement) 4. Cette recombinaison est stable et ne pas revenir 5. Un &quot;piste&quot; (LP) de séquence constitué par un gène de résistance à la spectinomycine-, aadA (SpR), et le E. coli ß-glucuronidase gène (uidA) flanqué par des sites attP a été intégré dans les chromosomes de Sinorhizobium meliloti, Ochrobactrum anthropi et Agrobacterium tumefaciens dans une région intergénique, l&#39;ampC locus, et le locus tetA, respectivement. S. meliloti est utilisé dans ce protocole. Vecteurs donateurs mobilisables contenant des sites attB flanquant un stuffer protéine fluorescente rouge (RFP) gène et un gène de résistance aux antibiotiques ont également été construits. Dans cet exemple la gentamicine pJH110 plasmide de résistance est utilisé. Le gène DP 6 peuvent être remplacés par une construction souhaité à l&#39;aide Sph I et Pst I. Sinon, une construction synthétique flanqué par des sites attB peut être sous-cloné dans un vecteur mobilisable comme pK19mob 7. L&#39;expression du gène de l&#39;intégrase ΦC31 (cloné à partir d&#39;pHS62 8) est entraîné par le promoteur lac, sur une large gamme d&#39;hôtes mobilisable plasmide pRK7813 9. Un protocole d&#39;accouplement tetraparental est utilisé pour transférer la cassette des bailleurs de fonds dans la souche LP remplaçant ainsi les marqueurs dans la séquence LP avec la cassette des bailleurs de fonds. Ces cellules sont trans-intégrants. Trans-intégrants sont formées avec un rendement typique de 0,5%. Trans-intégrants se trouvent généralement dans les premières colonies 500-1000 dépistées par la sensibilité aux antibiotiques ou bleu-blanc de dépistage utilisant le 5-bromo-4-chloro-3-indolyl-beta-D-acide glucuronique (X-gluc). Ce protocole contient les procédures d&#39;accouplement et de sélection pour la création et l&#39;isolement de trans-intégrants.

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)

Une méthode rapide et efficace d&#39;intégrer l&#39;ADN étranger dans des souches d&#39;intérêt accepteurs pré-faites, dites souches piste d&#39;atterrissage, est décrite. Procédé permettant d&#39;intégration spécifique de site d&#39;une cassette d&#39;ADN dans le locus piste d&#39;ingénierie d&#39;une souche donnée, par conjugaison et l&#39;expression de l&#39;intégrase ΦC31.

Site spécifique génie chromosome bactérien: ΦC31 intégrase médiation 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

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Bioengineering

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

15.5K vues.  University of Waterloo. Une méthode rapide et efficace d'intégrer l'ADN étranger dans des souches d'intérêt accepteurs pré-faites, dites souches piste d'atterrissage, est décrite. Procédé permettant d'intégration spécifique de site d'une cassette d'ADN dans le locus piste d'ingénierie d'une souche donnée, par conjugaison et l'expression de l'intégrase ΦC31.

Vidéo: 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.

Procédure pour l&#39;ingénierie du poumon

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

Recherche

Bio-ingénierie

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.

Caractériser Volatiles bactérienne secondaire à l&#39;aide d&#39;ionisation électrospray spectrométrie de masse (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).

Rendement élevé, spécifique au site transfection de cellules adhérentes avec l&#39;aide siARN réseaux de microélectrodes (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.

Capillaire de travail lithographie pour l&#39;ingénierie tissulaire cardiaque

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.

Observation et quantification du compactage du gel de fibrine médié par les fibroblastes

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.

Fabrication robuste préformes à fibres naturelles Utilisant bactérienne Cellulose 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.

Microbiologiquement Induced Précipitation de calcite Mediated par<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.

Génie tissulaire par Intrinsic vascularisation dans un<em&gt; In Vivo</em&gt; Tissue Chambre Ingénierie

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.

Microfluidique Buffer Exchange pour sans interférence Micro / Nanoparticules ingénierie cellulaire

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.

Formation et Patterning Light-médiée hydrogels pour la culture cellulaire Applications

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