Les auteurs tiennent à remercier le Dr Peiman Hemmati (Département de médecine, Université de Wisconsin-Madison) pour avoir généreusement fourni les MSC H1, et le Dr Tim Hacker, le Dr Song et Gouqing Mme Jill Koch de l&#39;Université du Wisconsin physiologie cardiovasculaire Facilité de base pour les opérations chirurgicales de la souris. Ce travail a été soutenu par la National Science Foundation grâce à une bourse de recherche supérieures à Brian Freeman et NIH R21 HL089679.

1. La transfection de la cellule donneuse <ol><li> Récolte des cellules souches mésenchymateuses (CSM, dérivée de cellules souches embryonnaires H1 fournie gracieusement par le Dr Peiman Hematti, ou alternativement, tout type de cellule d&#39;une espèce quelconque hypothèse de fusionner in vivo pourraient être employées) où 70 - 80% confluence avec 1X trypsine (Mediatech, Manassas VA) pendant 5 min. Désactiver la trypsine avec α-MEM milieu complet (antibiotique libre, Invitrogen, Carlsbad CA) 18. Centrifuger à 300 xg pendant 5 min. </li><li> Aspirer délicatement le surnageant et remettre en suspension le culot dans 1 ml de PBS 1X et compter les cellules en utilisant un hématimètre. </li><li> Transfert de 1,5 x 10 6 cellules dans un tube à centrifuger de 1,5 ml. Centrifuger à 300 xg pendant 5 min. </li><li> Aspirer délicatement le surnageant. Resuspendre le culot dans 300 ul de tampon R (système de transfection Néon, Invitrogen) et 6 mg (2 mg / 5,0 x 10 5 cellules) de P231 pCMVe-betaAc-STOP-Luc (Addgene, Cambridge, MA). Placer 3 ml de tampon E (InvitrOgen) dans le port d&#39;amarrage électroporation selon le protocole du fabricant (système de transfection Néon, Invitrogen). </li><li> Transfert cellulaire plasmide solution à un embout 100 Neon pipette ul et électroporation avec une durée d&#39;impulsion de 20 ms et d&#39;une magnitude de 1500 volts. Placez électroporées cellules dans un tube de 15 ml conique contenant 9,7 ml α-MEM milieu complet. </li><li> Répétez l&#39;étape 1.5 deux fois supplémentaires et les cellules transfectées piscine pour donner un volume total de 10 ml. Ajouter 10 ml de la suspension cellulaire (1,5 x 10 6 cellules) dans un flacon contenant 10 mL T175 α-MEM milieu complet. La viabilité des cellules après l&#39;électroporation est d&#39;environ 30%, pour donner environ 4,5 x 10 5 cellules viables par T175. </li><li> Changer α-MEM milieu complet 24 heures après transfection. </li><li> Récolte des cellules transfectées où 70 à 80% confluentes (~ 2 - 3 jours après l&#39;électroporation). Effectuer le nombre de cellules en utilisant un hématimètre et remettre les cellules à une concentration de 1,0 x 10 6cells/50 uL d&#39;α-MEM milieu complet. Minimisez le temps passer cellules en suspension afin de réduire la mort cellulaire avant l&#39;injection. </li></ol> 2. Injection intramyocardique <ol><li> Induire une anesthésie par isoflurane (Phoenix Pharmaceuticals, Inc, Saint Joseph, MO) sur des souris transgéniques conçues pour exprimer constitutivement la recombinase Cre dans chaque cellule (B6.C-Tg (CMV-cre) 1Cgn / J, Jackson Laboratory, Bar Harbor, ME). </li><li> Enlever les poils de la région de la poitrine à l&#39;aide tondeuses à cheveux ou épilateur chimique. </li><li> Intuber avec un cathéter de calibre 18 (Becton Dickinson &amp; Co, Franklin Lakes New Jersey) et les placer sur un ventilateur de souris à 120 à 130 respirations par minute avec un volume d&#39;éjection systolique de 150 uL. </li><li> Faire une incision latérale dans le quatrième espace intercostal, produisant ainsi une thoracotomie. </li><li> Visualisation du cœur, faire deux injections 25 pl de suspension de cellules transfectées avec un seringue de 1 mL (Temuro Medical Corporation, dans le Somerset, NJ) et une aiguille de calibre 28 (Bectsur Dickinson &amp; Co, Franklin Lakes New Jersey). Pour faciliter l&#39;injection intra-myocardique et prévenir des dommages excessifs à l&#39;orgue, plier la tête de l&#39;aiguille à 90 degrés. </li><li> Après l&#39;injection, l&#39;utilisation des sutures résorbables (par exemple, Vicryl) pour fermer les côtes et les couches musculaires. Suture cutanée fermée à l&#39;aide de nylon 4-0 ou en soie. </li><li> Laissez la souris pour récupérer de l&#39;anesthésie et l&#39;extubation. </li><li> Les groupes de contrôle devrait inclure des souris Cre recevant des injections moyennes seulement, les souris Cre recevoir la même concentration de cellules non transfectées et les souris de type sauvage recevant des cellules transfectées (alternativement, les souris recevant des cellules transfectées Cre pas enclin à fusibles). </li></ol> 3. D&#39;imagerie bio photonique <ol><li> Cinq à quinze minutes avant l&#39;imagerie, par voie intrapéritonéale (IP) d&#39;injecter 10 uL par gramme de poids corporel de souris de 15 mg / ml D-luciférine (Caliper Life Sciences, Hopkinton, MA). </li><li> Induire une anesthésie sur des souris via l&#39;isoflurane à 4% pour l&#39;induction uneD 1 - 2% pour la maintenance. </li><li> Placez la souris en position couchée dans la boîte de l&#39;imagerie avec un masque facial administrer 1 - 2% d&#39;isoflurane pour l&#39;anesthésie de maintenance (système d&#39;imagerie bio photonique de Xenogen, Hopkinton, MA). Plusieurs souris peuvent être imagés simultanément. La souris de contrôle d&#39;image avec des souris expérimentales simulacre pour faciliter la comparaison des signaux luminescents. </li><li> En utilisant le logiciel image vivante (Xenogen), mis le temps d&#39;exposition approprié (typiquement 60 sec, voir résultats). Réglez la zone de l&#39;image pour s&#39;adapter à des souris et de garder tout au long d&#39;imagerie zone cohérente pour empêcher toute modification de la sensibilité. Régler la hauteur de l&#39;objet à 4,5 cm. </li><li> Acquérir intensité du signal de luminescence correspondant à la souris ou des souris dans le champ de vision et d&#39;enregistrer des fichiers image non modifiée. Traiter les images de retirer le signal de fond correspondant à un contrôle ou une souris non manipulés. Les valeurs d&#39;intensité de fond ci-dessus correspondent aux cellules fusionnées au sein de l&#39;animal. Analyse de l&#39;intensité peut être effectuée à l&#39;aide du logiciel Image habitable (Xenogen) ou ouvrir le logiciel d&#39;analyse source de l&#39;image. Tabituellement, une région d&#39;intérêt (ROI) est sélectionnée pour comparer les données d&#39;intensité entre les animaux et les expériences. </li></ol> 4. Les résultats représentatifs Pour déterminer la sensibilité du système d&#39;imagerie biophotonique Xenogen, une lignée cellulaire qui exprime constitutivement la luciférase (LUC-231-D3H1, Xenogen) a été remis au myocarde de souris C57/BL6 (Jackson Laboratory). Les cellules ont été injectées à des concentrations de 1 x 10 6, 1 x 10 3, ou 1 x 10 cellules 1. Six heures après la livraison des cellules, les souris ont été injectées par voie intrapéritonéale avec luciférine et imagée en utilisant le système Xenogen. Un signal spécifique a pu être détecté avec 1000 cellules (2 de 6 souris imagé, figure 2), mais la détection est plus fiable avec 10 000 cellules (6 de 6 souris imagé). Surtout, cette étude a également servi à établir une corrélation grossière entre le nombre de cellules exprimant la luciférase et l&#39;intensité du signal. <p class="jove_content"&gt; Pour démontrer l&#39;utilité du protocole décrit dans la détection et le suivi de la fusion cellulaire, cellules souches mésenchymateuses ont été transfectées avec le plasmide LoxP-Stop-LoxP-luciférase (Addgene) et livrés au niveau du myocarde de Cre-exprimant les souris. Environ une semaine après la livraison des cellules, les souris ont d&#39;abord été imagées en utilisant le système Xenogen sans D-luciférine injection. Comme prévu, sans le substrat enzymatique, aucun signal d&#39;intensité a été détecté (Figure 3). Ensuite, D-luciférine a été injectée par voie intrapéritonéale et un signal correspondant à l&#39;intensité de la luciférase et donc la fusion cellulaire a été détecté dans deux des quatre souris testées. Un signal similaire a été détecté une semaine plus tard (figure 3), suggérant des produits de fusion MSC couplé peut être maintenue in vivo. Dans ce cas, l&#39;étude a été arrêtée pour permettre l&#39;évaluation du cœur et des tissus environnants, mais on pourrait envisager à plus long terme analyses et d&#39;imagerie plus fréquentes pour suivre l&#39;entretien, la prolifération uneD peut-être la migration des produits de fusion chez les souris. <img alt="Figure 1" src="/files/ftp_upload/3581/3581fig1.jpg" /> Figure 1. Schéma de la technique pour détecter la fusion cellulaire in vivo. Si la fusion entre les cellules exprimant la Cre-souris et des cellules transplantées exprimant un plasmide floxés luciférase survient, la luciférase sera exprimé. Luciférase peut être détectée par l&#39;injection du substrat enzymatique, D-luciférine, dans la souris et ensuite d&#39;imagerie de la souris en utilisant un système d&#39;imagerie biophotonique Xenogen (Adapté de 19) <img alt="Figure 2" src="/files/ftp_upload/3581/3581fig2.jpg" /> Figure 2. Sensibilité de la détection de cellules exprimant la luciférase dans le tissu cardiaque à l&#39;imagerie biophotonique. Une lignée cellulaire qui exprime constitutivement la luciférase (LUC-231-D3H1, Xenogen) a été livré à l&#39;espace intra-myocardique de souris C57/BL6 à différents nombre total de cellules. Des images représentatives de souris receiving 1 x 10 6, 1 x 10 3 et 1 x 10 1 cellules (de gauche à droite) sont présentées, l&#39;imagerie a été réalisée à environ 6 heures après l&#39;injection. <img alt="Figure 3" src="/files/ftp_upload/3581/3581fig3.jpg" /> Figure 3. Quantification de la luminescence Vivo indicatif de la fusion cellulaire. MSC ont été transfectées avec le LoxP-Stop-LoxP-luciférase plasmide et livrés à l&#39;myocarde de Cre-exprimant les souris. Environ une semaine et deux semaines après l&#39;accouchement de cellules, les souris Cre ont été imagées en utilisant le système d&#39;imagerie biophotonique Xenogen pour mesurer l&#39;intensité de la luminescence indicatif de la fusion cellulaire. (A) Superposition de la photographie et l&#39;intensité de la luminescence de l&#39;imposture et de souris 1-4 (de gauche à droite) jours après la livraison 17 cellules. (B) l&#39;intensité de la luminescence du simulacre et de souris 1-4 (de gauche à droite) jours après la livraison 17 cellules. Une région d&#39;intérêt a été sélectionné (jaune) correspondant au site d&#39;injection et l intensité deevels ont été déterminés en utilisant ImageJ (source libre) de logiciels 20. (C) Intensité de luminescence a été normalisée à la même région d&#39;intérêt sur le simulacre de la souris pour toutes les conditions expérimentales. À une semaine, les souris 3 et 4 montrent signal de luminescence positifs suggérant fusion spontanée d&#39;une cellule de la souris et le MSC transplantées. Le signal a persisté dans la souris 3 moins deux semaines. Pour déterminer la localisation des organes spécifiques du signal correspondant à la souris 3, la cavité thoracique a été exposé et les organes primaires excisée et imagée. (D) Superposition de la photographie et l&#39;intensité de la luminescence de la souris 3. Notez la localisation du signal d&#39;intensité dans l&#39;intestin grêle. (E) Intensité de luminescence de la souris 3. (F) Superposition de la photographie et l&#39;intensité de la luminescence de l&#39;imposture de la souris. (G) Intensité de luminescence du simulacre de la souris.

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Pas de conflits d&#39;intérêt déclarés.

La méthode décrite ici permet, pour la première fois, l&#39;identification et l&#39;analyse temporelle discrète de la fusion cellulaire dans les organismes, y compris les petits animaux. L&#39;approche combine Cre-LoxP de recombinaison avec l&#39;analyse d&#39;image ultérieures biophotonique. L&#39;approche est susceptible d&#39;être suivi non seulement fusion cellule-cellule, mais aussi fusion virus-cellule et ainsi pourraient se révéler utiles pour le suivi des infections virales. L&#39;analyse des images est ...

<table border="1"><tbody><tr><td> Nom du réactif </td><td> Société </td><td> Numéro de catalogue </td><td> Commentaires </td></tr><tr><td> Système de transfection Neon </td><td> Invitrogen, Carlsbad, CA </td><td> MPK5000 </td><td></td></tr><tr><td> Neon 100 Kit uL </td><td> Invitrogen, Carlsbad, CA </td><td> MPK10025 </td><td> Contient R et E Tampon </td></tr><tr><td> une-MEM en poudre </td><td> Invitrogen, Carlsbad, CA </td><td> 12000-022 </td><td></td></tr><tr><td> Sérum fœtal bovin (FBS) </td><td> Hyclone, Logan UT </td><td> SH30070.03 </td><td></td></tr><tr><td> B6.C-Tg (CMV-cre) 1Cgn / J </td><td> Jackson Laboratory, Bar Harbor, ME </td><td> 006054 </td><td></td></tr><tr><td> 10X Trypsine </td><td> Fisher Scientific, Forest Lawn, NJ </td&gt; <td> MT-25-054-Cl </td><td></td></tr><tr><td> L-glutamine </td><td> Fisher Scientific, Forest Lawn, NJ </td><td> 25030-081 </td><td></td></tr><tr><td> D-luciférine </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> 122796 </td><td></td></tr><tr><td> Système d&#39;imagerie bio photonique de Xenogen </td><td> Caliper Life Sciences, Hopkinton, MA </td><td> Spectre IVIS </td><td></td></tr><tr><td> Biocarbonate sodium </td><td> Sigma-Aldrich, St. Louis, MO </td><td> S6014-500G </td><td></td></tr><tr><td> Non essentiels Acides aminés </td><td> Invitrogen, Carlsbad, CA </td><td> 11140-050 </td><td></td></tr></tbody></table>

a cre-lox p recombination approach for the detection of cell fusion in vivo

La capacité de deux ou plusieurs cellules d&#39;un même type de fusionner a été utilisé chez les métazoaires long de l&#39;évolution de former de nombreux organes complexes, y compris le muscle squelettique, les os et le placenta. Les études contemporaines montrent la fusion de cellules du même type confère fonction améliorée. Par exemple, lorsque les cellules trophoblastiques du fusible du placenta pour former le syncytiotrophoblaste, le syncytiotrophoblaste est mieux en mesure de transporter les nutriments et les hormones à travers la barrière foeto-maternelle de trophoblastes non fusionnée 1-4. Des études plus récentes démontrent la fusion de cellules de différents types peuvent diriger le destin des cellules. Le &quot;retour&quot; ou la modification du destin cellulaire par fusion était autrefois considérée comme limitée aux systèmes de culture cellulaire. Mais l&#39;avènement de la transplantation de cellules souches conduit à la découverte par nous et d&#39;autres que les cellules souches peuvent fusionner avec les cellules somatiques in vivo et que la fusion facilite la différenciation des cellules souches 5-7. Ainsi, la fusion cellulaire est un processus réglementé capable de promouvoir la survie et la différenciation cellulaire et pourrait donc être d&#39;une importance centrale pour le développement, la réparation des tissus et même la pathogénie de la maladie. Limiter l&#39;étude de la fusion cellulaire, est le manque de technologie appropriée pour 1) identifier précisément les produits de fusion et à 2) des produits de fusion suivre dans le temps. Nous présentons ici une nouvelle approche pour répondre à la fois les limites via l&#39;induction de la bioluminescence lors de la fusion (figure 1);. Bioluminescence peuvent être détectés avec une haute sensibilité in vivo 8-15 Nous utilisons une construction codant pour la luciférase de luciole (Photinus pyralis) gène placé à côté de un codon stop flanqué de séquences LoxP. Lorsque les cellules exprimant ce gène avec fusible de cellules exprimant la protéine recombinase Cre, les sites loxP sont clivés et le signal d&#39;arrêt est excisé permettant la transcription de la luciférase. Parce que le signal est inducibles, l&#39;incidence des faux positifs signaux est très faible. Contrairement aux méthodes existantes qui utilisent le système Cre / LoxP 16, 17, nous avons intégré un signal «vivant» de détection et ainsi permettre pour la première fois la possibilité de suivre la cinétique de la fusion cellulaire in vivo. Afin de démontrer l&#39;approche, les souris exprimant la recombinase Cre omniprésent servi en tant que bénéficiaires des cellules souches transfectées avec une construction d&#39;exprimer la luciférase en aval d&#39;un codon stop floxés. Les cellules souches ont été transplantés par injection intra-myocardique et après analyse de transplantation d&#39;image intravitale a été menée pour vérifier la présence des produits de fusion dans le cœur et les tissus environnants au cours du temps. Cette approche pourrait être adaptée pour analyser la fusion cellulaire dans n&#39;importe quel type de tissu, à tout stade de développement, la maladie ou la réparation des tissus adultes.

Watch this Scientific Journal Video about A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo at JoVE.com

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Une méthode pour suivre la fusion cellulaire dans les organismes vivants au cours du temps est décrite. L&#39;approche utilise Cre-<em&gt; LoxP</emRecombinaison&gt; pour induire l&#39;expression de luciférase après la fusion cellulaire. Le signal luminescent généré peut être détecté dans les organismes vivants en utilisant des systèmes d&#39;imagerie bio photonique avec une sensibilité de détection de ~ 1000 cellules dans les tissus périphériques.

Une approche recombinaison Cre-Lox P pour la détection de la fusion cellulaire In Vivo</em

The ability of two or more cells of the same type to fuse has been utilized in metazoans throughout evolution to form many complex organs, including ...

a-cre-lox-p-recombination-approach-for-detection-cell-fusion

Research

JoVE Journal

Bioengineering

A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

18.7K vues.  University of Wisconsin-Madison. Une méthode pour suivre la fusion cellulaire dans les organismes vivants au cours du temps est décrite. L'approche utilise Cre- LoxP pour induire l'expression de luciférase après la fusion cellulaire. Le signal luminescent généré peut être détecté dans les organismes vivants en utilisant des systèmes d'imagerie bio photonique avec une sensibilité de détection de ~ 1000 cellules dans les tissus périphériques.

Vidéo: A Cre-Lox P Recombination Approach for the Detection of Cell Fusion In Vivo

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, environmental/agricultural/biodefense monitoring, nanobiotechnology, and more. For diagnostic applications, monitoring (detecting) the presence, absence, or abnormal expression of targeted proteomic or genomic biomarkers found in patient samples can be used to determine treatment approaches or therapy efficacy. In the research arena, information on molecular affinities and specificities are useful for fully characterizing the systems under investigation.
Many of the current systems employed to determine molecular concentrations or affinities rely on the use of labels. Examples of these systems include immunoassays such as the enzyme-linked immunosorbent assay (ELISA), polymerase chain reaction (PCR) techniques, gel electrophoresis assays, and mass spectrometry (MS). Generally, these labels are fluorescent, radiological, or colorimetric in nature and are directly or indirectly attached to the molecular target of interest. Though the use of labels is widely accepted and has some benefits, there are drawbacks which are stimulating the development of new label-free methods for measuring these interactions. These drawbacks include practical facets such as increased assay cost, reagent lifespan and usability, storage and safety concerns, wasted time and effort in labelling, and variability among the different reagents due to the labelling processes or labels themselves. On a scientific research basis, the use of these labels can also introduce difficulties such as concerns with effects on protein functionality/structure due to the presence of the attached labels and the inability to directly measure the interactions in real time.
Presented here is the use of a new label-free optical biosensor that is amenable to microarray studies, termed the Interferometric Reflectance Imaging Sensor (IRIS), for detecting proteins, DNA, antigenic material, whole pathogens (virions) and other biological material. The IRIS system has been demonstrated to have high sensitivity, precision, and reproducibility for different biomolecular interactions [1-3]. Benefits include multiplex imaging capacity, real time and endpoint measurement capabilities, and other high-throughput attributes such as reduced reagent consumption and a reduction in assay times. Additionally, the IRIS platform is simple to use, requires inexpensive equipment, and utilizes silicon-based solid phase assay components making it compatible with many contemporary surface chemistry approaches.
Here, we present the use of the IRIS system from preparation of probe arrays to incubation and measurement of target binding to analysis of the results in an endpoint format. The model system will be the capture of target antibodies which are specific for human serum albumin (HSA) on HSA-spotted substrates.

Biomolecular Detection employing the Interferometric Reflectance Imaging Sensor IRIS

Quantitative, high-throughput, real-time, and label-free biomolecular detection (DNA, protein, etc.) on SiO2 surfaces can be achieved using a simple interferometric technique which relies on LED illumination, minimal optical components, and a camera. The Interferometric Reflectance Imaging Sensor (IRIS) is inexpensive, simple to use, and amenable to microarray formats.

Détection biomoléculaire employant le capteur interférométrique Réflectance Imaging (IRIS)

The sensitive measurement of biomolecular interactions has use in many fields and industries such as basic biology and microbiology, ...

Recherche

Bio-ingénierie

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

La fabrication des électrochimique de l&#39;ADN des Biocapteurs pour la détection sans réactif d&#39;acides nucléiques, protéines et petites molécules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Creating Two-Dimensional Patterned Substrates for Protein and Cell Confinement

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1 While microcontact printing can be employed to directly print a large number of molecules, including proteins,2 DNA,3 and silanes,4 the formation of self-assembled monolayers (SAMs) from long chain alkane thiols on gold provides a simple way to confine proteins and cells to specific patterns containing adhesive and resistant regions. This confinement can be used to control cell morphology and is useful for examining a variety of questions in protein and cell biology. Here, we describe a general method for the creation of well-defined protein patterns for cellular studies.5 This process involves three steps: the production of a patterned master using photolithography, the creation of a PDMS stamp, and microcontact printing of a gold-coated substrate. Once patterned, these cell culture substrates are capable of confining proteins and/or cells (primary cells or cell lines) to the pattern.
The use of self-assembled monolayer chemistry allows for precise control over the patterned protein/cell adhesive regions and non-adhesive regions; this cannot be achieved using direct protein stamping. Hexadecanethiol, the long chain alkane thiol used in the microcontact printing step, produces a hydrophobic surface that readily adsorbs protein from solution. The glycol-terminated thiol, used for backfilling the non-printed regions of the substrate, creates a monolayer that is resistant to protein adsorption and therefore cell growth.6 These thiol monomers produce highly structured monolayers that precisely define regions of the substrate that can support protein adsorption and cell growth. As a result, these substrates are useful for a wide variety of applications from the study of intercellular behavior7 to the creation of microelectronics.8
While other types of monolayer chemistry have been used for cell culture studies, including work from our group using trichlorosilanes to create patterns directly on glass substrates,9 patterned monolayers formed from alkane thiols on gold are straight-forward to prepare. Moreover, the monomers used for monolayer preparation are commercially available, stable, and do not require storage or handling under inert atmosphere. Patterned substrates prepared from alkane thiols can also be recycled and reused several times, maintaining cell confinement.10

Self-assembled monolayers (SAMs) formed from long chain alkane thiols on gold provide well-defined substrates for the formation of protein patterns and cell confinement. Microcontact printing of hexadecanethiol using a polydimethylsiloxane (PDMS) stamp followed by backfilling with a glycol-terminated alkane thiol monomer produces a pattern where protein and cells adsorb only to the stamped hexadecanethiol region.

Créer deux dimensions Substrats motifs pour le confinement des protéines et de cellules

Microcontact printing provides a rapid, highly reproducible method for the creation of well-defined patterned substrates.1  While microcontact printing ...

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are available for microfluidic devices using soft lithography techniques 1-3. Microfluidic devices have been used for sub-cellular imaging 4,5, in vivo laser microsurgery 2,6 and cellular imaging 4,7. In vivo imaging requires immobilization of organisms. This has been achieved using suction 5,8, tapered channels 6,7,9, deformable membranes 2-4,10, suction with additional cooling 5, anesthetic gas 11, temperature sensitive gels 12, cyanoacrylate glue 13 and anesthetics such as levamisole 14,15. Commonly used anesthetics influence synaptic transmission 16,17 and are known to have detrimental effects on sub-cellular neuronal transport 4. In this study we demonstrate a membrane based poly-dimethyl-siloxane (PDMS) device that allows anesthetic free immobilization of intact genetic model organisms such as Caenorhabditis elegans (C. elegans), Drosophila larvae and zebrafish larvae. These model organisms are suitable for in vivo studies in microfluidic devices because of their small diameters and optically transparent or translucent bodies. Body diameters range from ~10 &mu;m to ~800 &mu;m for early larval stages of C. elegans and zebrafish larvae and require microfluidic devices of different sizes to achieve complete immobilization for high resolution time-lapse imaging. These organisms are immobilized using pressure applied by compressed nitrogen gas through a liquid column and imaged using an inverted microscope. Animals released from the trap return to normal locomotion within 10 min.
We demonstrate four applications of time-lapse imaging in C. elegans namely, imaging mitochondrial transport in neurons, pre-synaptic vesicle transport in a transport-defective mutant, glutamate receptor transport and Q neuroblast cell division. Data obtained from such movies show that microfluidic immobilization is a useful and accurate means of acquiring in vivo data of cellular and sub-cellular events when compared to anesthetized animals (Figure 1J and 3C-F 4).
Device dimensions were altered to allow time-lapse imaging of different stages of C. elegans, first instar Drosophila larvae and zebrafish larvae. Transport of vesicles marked with synaptotagmin tagged with GFP (syt.eGFP) in sensory neurons shows directed motion of synaptic vesicle markers expressed in cholinergic sensory neurons in intact first instar Drosophila larvae. A similar device has been used to carry out time-lapse imaging of heartbeat in ~30 hr post fertilization (hpf) zebrafish larvae. These data show that the simple devices we have developed can be applied to a variety of model systems to study several cell biological and developmental phenomena in vivo.

Simple Microfluidic Devices for in vivo Imaging of C. elegans, Drosophila and Zebrafish

A simple microfluidic device has been developed to perform anesthetic free in vivo imaging of C. elegans, intact Drosophila larvae and zebrafish larvae. The device utilizes a deformable PDMS membrane to immobilize these model organisms in order to perform time lapse imaging of numerous processes such as heart beat, cell division and sub-cellular neuronal transport. We demonstrate the use of this device and show examples of different types of data collected from different model systems.

Simple Dispositifs microfluidiques pour<em&gt; In vivo</em&gt; Imagerie des<em&gt; C. elegans</em&gt;,<em&gt; Drosophila</em&gt; Et le poisson-zèbre

Micro fabricated fluidic devices provide an accessible micro-environment for in vivo studies on small organisms. Simple fabrication processes are ...

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to maintain cellular homeostasis. Puncture injury with glass needles provides a direct method to trigger a rapid epidermal wound response that activates wound transcriptional reporters, which can be visualized by a localized reporter signal in living embryos or larvae. Puncture or laser injury also provides signals that promote the recruitment of hemocytes to the wound site. Surprisingly, severe (through and through) puncture injury in late stage embryos only rarely disrupts normal embryonic development, as greater than 90% of such wounded embryos survive to adulthood when embryos are injected in an oil medium that minimizes immediate leakage of hemolymph from puncture sites. The wound procedure does require micromanipulation of the Drosophila embryos, including manual alignment of the embryos on agar plates and transfer of the aligned embryos to microscope slides. The Drosophila epidermal wound response assay provides a quick system to test the genetic requirements of a variety of biological functions that promote wound healing, as well as a way to screen for potential chemical compounds that promote wound healing. The short life cycle and easy culturing routine make Drosophila a powerful model organism. Drosophila clean wound healing appears to coordinate the epidermal regenerative response, with the innate immune response, in ways that are still under investigation, which provides an excellent system to find conserved regulatory mechanisms common to Drosophila and mammalian epidermal wounding.

Microinjection Wound Assay and In vivo Localization of Epidermal Wound Response Reporters in Drosophila Embryos.

The embryonic epidermis of very late stage Drosophila embryos provides an in vivo system for rapid puncture wound response analysis and can be combined with genetic manipulations or chemical microinjection treatments to advance studies in wound healing for translation into mammalian models.

Microinjection plaies dosage et<em&gt; In vivo</em&gt; Localisation des épidermique plaies Reporters réponse dans<em&gt; Drosophila</em&gt; Embryons.

The Drosophila embryo develops a robust epidermal layer that serves both to protect the internal cells from a harsh external environment as well as to ...

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to evaluate new strategies that aim at improving vascularization, reliable methods are required to make the in-growth of new blood vessels into bio-artificial scaffolds visible and quantify the results. Over the past couple of years, our group has introduced a full skin defect model that enables the direct visualization of blood vessels by transillumination and provides the possibility of quantification through digital segmentation. In this model, one surgically creates full skin defects in the back of mice and replaces them with the material tested. Molecules or cells of interest can also be incorporated in such materials to study their potential effect. After an observation time of one&#8217;s own choice, materials are explanted for evaluation. Bilateral wounds provide the possibility of making internal comparisons that minimize artifacts among individuals as well as of decreasing the number of animals needed for the study. In comparison to other approaches, our method offers a simple, reliable and cost effective analysis. We have implemented this model as a routine tool to perform high-resolution screening when testing vascularization of different biomaterials and bio-activation approaches.

A Full Skin Defect Model to Evaluate Vascularization of Biomaterials In Vivo

Vascularization is key to approaches in successful tissue engineering. Therefore, reliable technologies are required to evaluate the development of vascular networks in tissue-constructs. Here we present a simple and cost-effective method to visualize and quantify vascularization in vivo.

Une peau pleine de défauts modèle pour évaluer la vascularisation des biomatériaux<em&gt; In Vivo</em

Insufficient vascularization is considered to be one of the main factors limiting the clinical success of tissue-engineered constructs. In order to ...

A Label-free Technique for the Spatio-temporal Imaging of Single Cell Secretions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning of such signaling can lead to many disorders. To understand cell-to-cell signaling, it is essential to study the spatial and temporal nature of the secreted molecules from the cell without disturbing the local environment. Various assays have been developed to study protein secretion, however, these methods are typically based on fluorescent probes which disrupt the relevant signaling pathways. To overcome this limitation, a label-free technique is required.
In this paper, we describe the fabrication and application of a label-free localized surface plasmon resonance imaging (LSPRi) technology capable of detecting protein secretions from a single cell. The plasmonic nanostructures are lithographically patterned onto a standard glass coverslip and can be excited using visible light on commercially available light microscopes. Only a small fraction of the coverslip is covered by the nanostructures and hence this technique is well suited for combining common techniques such as fluorescence and bright-field imaging.
A multidisciplinary approach is used in this protocol which incorporates sensor nanofabrication and subsequent biofunctionalization, binding kinetics characterization of ligand and analyte, the integration of the chip and live cells, and the analysis of the measured signal. As a whole, this technology enables a general label-free approach towards mapping cellular secretions and correlating them with the responses of nearby cells.

Inter-cellular communication is critical for controlling various physiological activities within and outside the cell. This paper describes a protocol for measuring the spatio-temporal nature of single cell secretions. To achieve this, a multidisciplinary approach is used which integrates label-free nanoplasmonic sensing with live cell imaging.

Une technique Label-free pour l&#39;imagerie spatio-temporelle de cellule unique sécrétions

Inter-cellular communication is an integral part of a complex system that helps in maintaining basic cellular activities. As a result, the malfunctioning ...

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage dependent cells on the 2-dimensional surface of tissue culture plastic. More recently, there is a growing body of data demonstrating more in vivo-like behaviors of cells grown in 3-dimensional culture systems. This manuscript describes in detail the set-up and operation of a hollow fiber bioreactor system for the in vivo-like culture of mammalian cells. The hollow fiber bioreactor system delivers media to the cells in a manner akin to the delivery of blood through the capillary networks in vivo. The system is designed to fit onto the shelf of a standard CO2 incubator and is simple enough to be set-up by any competent cell biologist with a good understanding of aseptic technique. The systems utility is demonstrated by culturing the hepatocarcinoma cell line HepG2/C3A for 7 days. Further to this and in line with other published reports on the functionality of cells grown in 3-dimensional culture systems the cells are shown to possess increased albumin production (an important hepatic function) when compared to standard 2-dimensional tissue culture.

Hollow Fiber Bioreactors for In Vivo-like Mammalian Tissue Culture

The functional behavior of cells in culture can be improved by culturing in more in vivo-like 3-dimensional culture environments16-21. This manuscript describes the set-up and operation of a hollow fiber bioreactor system for in vivo-like mammalian tissue culture.

Bioréacteurs à fibres creuses pour<em&gt; In Vivo</em&gt; -like Tissue Culture Mammifères

Tissue culture has been used for over 100 years to study cells and responses ex vivo. The convention of this technique is the growth of anchorage ...

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Historically, most cellular processes have been studied in only 2 dimensions. While these studies have been informative about general cell signaling mechanisms, they neglect important cellular cues received from the structural and mechanical properties of the local microenvironment and extracellular matrix (ECM). To understand how cells interact within a physiological ECM, it is important to study them in the context of 3 dimensional assays. Cell migration, cell differentiation, and cell proliferation are only a few processes that have been shown to be impacted by local changes in the mechanical properties of a 3-dimensional ECM. Collagen I, a core fibrillar component of the ECM, is more than a simple structural element of a tissue. Under normal conditions, mechanical cues from the collagen network direct morphogenesis and maintain cellular structures. In diseased microenvironments, such as the tumor microenvironment, the collagen network is often dramatically remodeled, demonstrating altered composition, enhanced deposition and altered fiber organization. In breast cancer, the degree of fiber alignment is important, as an increase in aligned fibers perpendicular to the tumor boundary has been correlated to poorer patient prognosis1. Aligned collagen matrices result in increased dissemination of tumor cells via persistent migration2,3. The following is a simple protocol for embedding cells within a 3-dimensional, fibrillar collagen hydrogel. This protocol is readily adaptable to many platforms, and can reproducibly generate both aligned and random collagen matrices for investigation of cell migration, cell division, and other cellular processes in a tunable, 3-dimensional, physiological microenvironment.

Preparation of 3D Collagen Gels and Microchannels for the Study of 3D Interactions In Vivo

Collagen is a core component of the ECM, and provides essential cues for several cellular processes ranging from migration to differentiation and proliferation. Provided here is a protocol for embedding cells within 3D collagen hydrogels, and a more advanced technique for generating randomized or aligned collagen matrices using PDMS microchannels.

Préparation de la 3D collagène Gels et microcanaux pour l&#39;étude des interactions 3D<em&gt; In Vivo</em

Historically, most cellular processes have been studied in only 2 dimensions.  While these studies have been informative about general cell signaling ...

Visual Detection of Multiple Nucleic Acids in a Capillary Array

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.

This protocol describes the fabrication of a small, ready-to-use cassette that can be applied for visual detection of multiple nucleic acids in a single, test that is easy to operate. In this approach, a capillary array was used for multiplex and highly efficient detection of GMO targets.

Détection visuelle de plusieurs acides nucléiques dans une gamme capillaire

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently ...