Les auteurs remercient Ronald F. Renzi et Michael S. Bartsch pour leur contribution à la conception et au développement de dispositifs DMF et le moyeu DMF. Cette recherche a été entièrement pris en charge par le Laboratoire de recherche en scène et le programme de développement au Sandia National Laboratories. Sandia est un laboratoire multi-programme géré et exploité par Sandia Corporation, une filiale en propriété exclusive de Lockheed Martin Corporation, pour le ministère de l&#39;Administration nationale de sécurité nucléaire américain de l&#39;Énergie sous contrat DE-AC04-94AL85000.

Ce protocole général est conçu pour appuyer l&#39;application de la plate-forme pour un large éventail d&#39;études, les aspects spécifiques à l&#39;étude représentative décrite dans le présent rapport sont séparés entre parenthèses figure 2 illustre une étude représentative réalisée à l&#39;aide du protocole.. Notez que dans le protocole, toutes les commandes &quot;instruire&quot; sont automatisées grâce à l&#39;utilisation de scripts préétablis. Notez aussi que l&#39;étape 2 peut être effectuée en parallèle avec l&#39;étape 1 (par exemple pendant les étapes 1.7 et 1.8), et que l&#39;étape 2.1 est souvent contournés (car les plaques de l&#39;appareil DMF motifs / revêtement peuvent être lavés et réutilisés; voir l&#39;étape 5.2) . 1. Assembler et Remplir les Chambres de perfusion portables <ol><li> Enduire les surfaces intérieures de stérilisation (autoclave) microcapillaires (silice fondue; 679 um od, 534 Identifiant du um; 15 cm de long) avec la fibronectine, en demandant à la pompe à seringue pour aspirer une solution de fibronectine stérile (30 pl de 50 pg / ml) dans chaquemicrocapillaires, l&#39;incubation (37 ° C pendant 2 heures), ordonnant la pompe à seringue pour prélever la solution, et en laissant le microcapillaires sec (température ambiante (RT) pendant 6 heures). </li><li> Connecter chaque microcapillaire fibronectine revêtu (chambre de perfusion) à un tube de polycarbonate (500 um de diamètre extérieur, 100 ° du um) et de la tubulure de la pompe à seringue à ports multiples, utilisant des raccords CapTite (6 chambres de perfusion, 8 ports pompe de seringue). </li><li> Utilisation de ruban adhésif, fixer le corps de chaque chambre de perfusion à un bloc de chaleur fixé à la température désirée (37 ° C). </li><li> Plonger les extrémités ouvertes des chambres de perfusion à un réservoir (tube de microcentifuge ou une plaque de microtitrage) des cellules contenant (P388D.1 macrophages murins) en suspension dans du milieu de croissance frais (milieu RPMI-1640, 10% FBS, 500 unités / ml de pénicilline, 500 ug / ml de streptomycine) à une concentration souhaitée (10 6 cellules / ml). </li><li> Demandez à la pompe de la seringue pour dessiner des cellules (10 ul, 10 4 cellules) dans chaque chambre de perfusion suivie d&#39;assez intérimr pour déplacer le bouchon de liquide (~ 4,5 cm) de la partie solidaire du bloc de chauffage. </li><li> Permettre aux cellules d&#39;adhérer aux surfaces intérieures revêtues de fibronectine des chambres de perfusion (37 ° C pendant 1 - 2 h). Dans l&#39;intervalle, transférer les extrémités ouvertes des chambres de perfusion à partir du réservoir de cellules à un nouveau réservoir contenant un milieu de croissance frais. </li><li> Après la période d&#39;adhésion, charger le pousse-seringue pour envoyer les bouchons liquides (milieux conditionnés et les cellules seules) à perdre, retirer du milieu frais (10 pi), et suivre avec assez d&#39;air pour positionner les nouveaux bouchons liquides (milieu frais) sur la adhéré cellules. </li><li> Continuer à mener des échanges moyens (c.-à répéter l&#39;étape 1.7) à intervalles réguliers (toutes les 2 h) jusqu&#39;à ce que les populations cellulaires sont suffisamment équilibrée et étendue (16 à 24 h de culture à petite échelle des populations d&#39;affaires de ~ 1000 cellules / chambre). </li></ol> 2. Fabriquer le dispositif de DMF et assembler l&#39;architecture Hub <ol><li&gt; comme décrit précédemment 14,29, modèle de la plaque inférieure de l&#39;appareil DMF avec quarante-six oxyde d&#39;indium-étain (ITO) des électrodes (conducteurs de gouttelettes actionnement) par photolithographie et gravure, et les enrober de 5 microns de Parylene-C et 50 nm de Téflon AF. Former la plaque de dessus du dispositif de DMF par revêtement d&#39;un substrat de verre sans dessin ITO avec de Téflon AF, comme ci-dessus. </li><li> L&#39;utilisation des trames de compression en métal, fixer les plaques de DMF dans un moule polymère 14,30 avec des évidements qui maintiennent un espacement de 400 pm entre les plaques; cet espacement, combinée avec la taille de l&#39;électrode d&#39;actionnement (2,5 mm 2), qui définit le volume de la gouttelette (2,5 pl par électrode). Notez que l&#39;assemblage DMF peut être accompli par des moyens plus simples 24. </li><li> Insert revêtu de Téflon transfert microcapillaires (360 um de diamètre extérieur, 100 ° du um; 3,5 à 4,0 cm de long) dans l&#39;espace entre les plaques DMF, le positionnement de chaque pour s&#39;étendre vers le bord de l&#39;électrode d&#39;actionnement apparenté. </li><li> À la salle opposéd de chaque transfert apposer microcapillaires un raccord CapTite. </li><li> Engager les connecteurs électriques pour fournir une tension aux électrodes d&#39;ITO. séquences d&#39;activation des électrodes sont automatisées grâce à l&#39;utilisation d&#39;une interface électronique commandé par ordinateur exécutant des scripts préétablis. </li><li> En option: Déplacer la plaque tournante du DMF assemblé sur la scène de translation d&#39;un microscope (SZ-6145TR (Olympus, Japon)) équipé d&#39;un dispositif à couplage de charge (CCD) (DCR-HC96 (Sony, Japon)) pour faciliter le suivi de gouttelettes 31. </li></ol> 3. Relier les chambres de perfusion au Hub DMF et stimuler les populations cellulaires <ol><li> Retirez les extrémités ouvertes des chambres de perfusion du réservoir de fluide (voir l&#39;étape 1.6) et insérez-les dans les raccords CapTite fixés aux extrémités du transfert microcapillaires du moyeu DMF (voir l&#39;étape 2.4). </li><li> Demandez à la pompe de la seringue pour envoyer les bouchons liquides (milieux conditionnés) dans les chambres de perfusion à perdre. </li><li> InstRUCT le moyeu du DMF pour dessiner stimulus (E. coli particules biologiques à 100 pg / ml dans du milieu frais contenant 0,1% de Pluronic F127 p / v) à partir d&#39;un réservoir embarqué et délivrer une gouttelette de taille appropriée (10 pl) pour chaque transfert microcapillaire . Les réservoirs de bord sont composés de compartiments de forme cylindrique (1,5 x 1,5 cm chacune) et reliée au moyeu par l&#39;intermédiaire de tubes de DMF. </li><li> Demandez à la pompe à seringue pour aspirer le stimulus se branche sur le transfert microcapillaires, et suivre avec assez d&#39;air pour positionner les bouchons sur les populations de cellules dans les chambres de perfusion. </li><li> Incuber les cellules avec des mesures de relance (37 ° C pendant 1-4 heures). Facultatif: Échange de relance frais (c. répétez les étapes 3,2-3,4) à intervalles réguliers. </li></ol> 4. Mettre fin à la stimulation et récupérer Lysats cellulaires pour l&#39;analyse <ol><li> Facultatif: si une période post-stimulation est nécessaire, échanger les bouchons liquides stimulus contenant de milieu frais (c. répétez les étapes 3,2 à 3.4, En remplaçant un milieu frais de relance), et continuer à incuber et échanger si nécessaire. </li><li> Echange les bouchons liquide dans les chambres de perfusion pour le tampon de lavage (Prelude Direct Lysis Buffer Module B) (c. répétez les étapes 3.2 à 3.4, en remplaçant le tampon de lavage de relance), et incuber au besoin (5 min). </li><li> Bourse Les bouchons liquides (tampon de lavage) pour la solution de lyse (Prelude Direct tampon du module de lyse A avec 0,1% de Pluronic F127 w / v) (c. répétez les étapes 3.2 à 3.4, en remplaçant la solution de lyse de relance), et incuber au besoin (5 min) . </li><li> Demandez à la pompe de la seringue pour envoyer les bouchons liquides (lysats cellulaires) au moyeu DMF. </li><li> Démonter le moyeu DMF, retirez la plaque supérieure de l&#39;appareil DMF (en prenant soin de ne pas incliner la plaque de côté, car cela peut entraîner des gouttelettes de mélange), et de recueillir les lysats en utilisant un Pipetman ou une seringue pour une analyse hors-plate-forme (profilage de l&#39;expression génique par qPCR). </li></ol> 5. Propre plate-forme matérielle pourRéutiliser <ol><li> Plonger le transfert microcapillaires et raccords CapTite en Javel à 10% (v / v dans l&#39;eau) pendant 10 min à température ambiante, bien rincer avec de l&#39;eau déminéralisée et sec en utilisant de l&#39;azote gazeux. </li><li> Plonger les plaques d&#39;appareils DMF en Javel à 10% pendant 10 min à température ambiante, bien rincer avec de l&#39;isopropanol puis de l&#39;eau déminéralisée et sec en utilisant de l&#39;azote gazeux suivie d&#39;une cuisson à 160 ° C pendant 10 min. </li><li> Le tube de polycarbonate de la seringue pompe, et la fonte de polymère de l&#39;appareil DMF et le cadre de compression, peut être réutilisée sans nettoyage. La perfusion chambre microcapillaires sont jetés après chaque expérience. </li></ol>

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H., Oliver, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microarrays,+deep+sequencing+and+the+true+measure+of+the+transcriptome.">Microarrays, deep sequencing and the true measure of the transcriptome.</a> BMC Biology. 9, (2011).</li><li>Wu, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidically-unified+cell+culture,+sample+preparation,+imaging+and+flow+cytometry+for+measurement+of+cell+signaling+pathways+with+single+cell+resolution.">Microfluidically-unified cell culture, sample preparation, imaging and flow cytometry for measurement of cell signaling pathways with single cell resolution.</a> Lab on a Chip. 12, 2823-2831 (2012).</li><li>Srivastava, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+integrated+microfluidic+platform+enabling+automated+phosphoprofiling+of+macrophage+response.">Fully integrated microfluidic platform enabling automated phosphoprofiling of macrophage response.</a> Analytical Chemistry. 81, 3261-3269 (2009).</li><li>Herr, A. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microfluidic+immunoassays+as+rapid+saliva-based+clinical+diagnostics.">Microfluidic immunoassays as rapid saliva-based clinical diagnostics.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 5268-5273 (2007).</li></ol>

Les auteurs déclarent qu&#39;ils n&#39;ont aucun intérêt financier concurrents.

Nous avons développé une plate-forme automatisée simple et polyvalent pour la culture cellulaire à micro-échelle et des expériences de stimulation. La plate-forme nous permet de travailler avec de petits volumes de culture et les populations cellulaires (1 - 20 pi et 100 - 2.000 cellules, par chambre); dimensions de la culture pourrait être encore réduit par l&#39;utilisation de micro-capillaires de petit diamètre. Travailler à ces échelles de réduire le coût des études de routine et fait des étu...

L&#39;étude des cellules maintenues en culture (analyse in vitro) a fourni de précieuses informations sur les principes fondamentaux et les mécanismes moléculaires qui régissent les systèmes biologiques complexes et la santé humaine. Les méthodes conventionnelles de culture, la stimulation et la collecte de cellules d&#39;analyse, qui utilisent des boîtes de Pétri et les plaques de microtitration, ont été conçus pour l&#39;étude de grandes populations de cellules (≥ 10 5) dans des volumes de culture millilitre échelle (≥ 0,1 ml). Cependant, il existe de nombreux cas où des quantités limitées de cellules sont disponibles (par exemple des cellules primaires), ou de petites populations de cellules sont souhaitables (par exemple pour réduire la variabilité de cellule à cellule dans la population), ou réactifs nécessaires sont difficiles à obtenir ou trop coûteux (par exemple les facteurs cellulaires sécrétées purifiées). Ces problèmes peuvent être résolus avec succès par révision à la baisse la taille de la culture, qui a l&#39;avantage supplémentaire de réduire la consommation de tous lesréactifs nécessaires à l&#39;analyse 1,2 vitro. Malheureusement, le matériel et les méthodes conventionnelles ne supportent pas une manipulation précise et reproductible des cultures de micro-échelle et les systèmes automatisés microfluidique à base actuellement disponibles 3-11 sont trop complexes et spécialisés pour une utilisation de routine par la plupart des laboratoires. Dans ce rapport, nous décrivons l&#39;assemblage et l&#39;utilisation d&#39;une plate-forme technologique simple et polyvalent pour la culture automatisé, la stimulation et la récupération de petites populations de cellules (100 - 2.000 cellules) dans des volumes micro-échelle (1 - 20 pi). L&#39;architecture de la plate-forme (figure 1) est de conception modulaire: un ensemble de fibronectine revêtu microcapillaires («chambres de perfusion de cellules&quot; du module) sert de site pour l&#39;établissement, l&#39;entretien et la stimulation des cultures de micro-échelle et une microfluidique numérique (DMF ) 12,13 appareil équipé de &quot;transfert&quot; microcapillaires (module &quot;central hub&quot;) 14,15 Cellules itinéraireset des réactifs vers et à partir des chambres de perfusion. DMF permet à l&#39;utilisateur de régler individuellement plusieurs gouttelettes simultanément et modifier ou réorganiser les manipulations (c. trains de traitement des échantillons de reconfigurer) sans altérer le matériel de l&#39;appareil. Sa grande flexibilité est évidente dans sa récente apparition comme une technologie clé dans un large éventail d&#39;applications, y compris la culture de cellules 16,17, 18,19 dosages enzymatiques, immunologiques 20,21, 22,23 analyse de l&#39;ADN, la transformation des protéines, 24,25 et le traitement des échantillons cliniques. 26,27 Notre hub central tire parti de la flexibilité inhérente aux dispositifs DMF, et améliore encore ce grâce à l&#39;ajout d&#39;interfaces microcapillaires, qui offre la possibilité de réaliser un sous-ensemble de manipulations (par exemple, la culture de cellules) dans les zones périphériques spécialisés modules, plutôt que sur l&#39;appareil lui-même DMF. Cloisonnement des trains de traitement de cette manière simplifie également la conception de l&#39;APLtform architecture (pas besoin de construire un dispositif de DMF qui peut effectuer toutes les étapes de transformation) et facilite son évolution en tant que de nouvelles fonctions sont nécessaires (il suffit d&#39;intégrer de nouveaux modules périphériques que nécessaire). Transport des cellules et des réactifs à l&#39;intérieur du moyeu central est entraîné par électromouillage forces engendrées par l&#39;activation séquentielle des électrodes à l&#39;intérieur du dispositif DMF 13,28, le transport à destination, et à l&#39;intérieur des chambres de perfusion est effectuée par des changements de pression générés par une seringue à haute précision pompe. Tous ces mouvements fluides sont contrôlés via une interface électronique simple et automatisée grâce à l&#39;utilisation de scripts préétablis. Comme un exemple représentatif, nous démontrons l&#39;utilisation de la plate-forme pour l&#39;étude des réponses transcriptionnelles suscité dans les cellules immunitaires à défi avec des bactéries (Figure 2). La réalisation de ces expériences sur la plate-forme nous a permis de travailler avec de petits nombres de cellules (~ 1000 par expérimentationétat mental), minimiser la variabilité expérience à l&#39;expérience, les réactifs de conserver et re-direct-mains sur le travail. Compte tenu des avantages qu&#39;il confère, ainsi que son accessibilité et polyvalence, cette plate-forme devrait trouver une utilisation dans une grande variété de laboratoires et d&#39;applications et se révéler particulièrement utile pour faciliter l&#39;analyse des cellules et des stimuli qui sont disponibles en quantités limitées.

<table border="1">
 <tbody>
 <tr>
 <td>Name of Reagent/Material</td>
 <td>Company</td>
 <td>Catalog Number</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>Prelude Direct Lysis Module</td>
 <td>NuGEN</td>
 <td>1400-24</td>
 <td></td>
 </tr>
 <tr>
 <td>Trypan Blue (0.4% w/v)</td>
 <td>GIBCO</td>
 <td>15250-061</td>
 <td></td>
 </tr>
 <tr>
 <td>Cell Stripper</td>
 <td>Cellgro</td>
 <td>25-056-C1</td>
 <td></td>
 </tr>
 <tr>
 <td>Ovation PicoSL WTA</td>
 <td>NuGEN</td>
 <td>3310-048</td>
 <td></td>
 </tr>
 <tr>
 <td>Agencourt RNAClean XP</td>
 <td>Beckman Coulter Genomics</td>
 <td>A63987</td>
 <td></td>
 </tr>
 <tr>
 <td>pHrodo BioParticles</td>
 <td>Invitrogen</td>
 <td>P35361</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL4 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443111_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>CCL5 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm01302428_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>PTGS2 TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00478374_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>TNF TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm00443258_m1</td>
 <td></td>
 </tr>
 <tr>
 <td>GAPDH TaqMan qRT-PCR assay</td>
 <td>Applied Biosystems</td>
 <td>Mm99999915_g1</td>
 <td></td>
 </tr>
 <tr>
 <td>Pluronic F127</td>
 <td>Sigma Chemical</td>
 <td>2594628</td>
 <td></td>
 </tr>
 <tr>
 <td>Fluorinert FC-40</td>
 <td>Sigma Chemical</td>
 <td>51142-49-5</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene C dimer</td>
 <td>Specialty Coating Systems</td>
 <td>28804-46-8</td>
 <td></td>
 </tr>
 <tr>
 <td>Teflon-AF</td>
 <td>DuPont</td>
 <td>AF1600</td>
 <td></td>
 </tr>
 <tr>
 <td>Polyimide tape</td>
 <td>ULINE</td>
 <td>S-11928</td>
 <td></td>
 </tr>
 <tr>
 <td>Indium tin oxide (ITO) coated glass substrates </td>
 <td>Delta Technologies </td>
 <td>CB-40IN-1107</td>
 <td></td>
 </tr>
 <tr>
 <td>DMF hub Teflon-coated fused-silica microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSU100375</td>
 <td></td>
 </tr>
 <tr>
 <td>Perfusion chamber microcapillaries</td>
 <td>Polymicro Technologies</td>
 <td>TSP530700</td>
 <td></td>
 </tr>
 <tr>
 <td>Tubing and microcapillary fittings</td>
 <td>Sandia National Laboratories</td>
 <td>&nbsp;</td>
 <td></td>
 </tr>
 <tr>
 <td>Polycarbonate tubing</td>
 <td>Paradigm Optics</td>
 <td>CTPC100-500-5</td>
 <td></td>
 </tr>
 <tr>
 <td>8-port precision syringe pump equipped with 30 mm (500 &mu;l capacity) syringes</td>
 <td>Hamilton Company</td>
 <td>54848-01</td>
 <td></td>
 </tr>
 <tr>
 <td>Parylene-C vapor deposition instrument</td>
 <td>Specialty Coating Systems</td>
 <td>PDS 2010 Labcoter 2</td>
 <td></td>
 </tr>
 <tr>
 <td>High-voltage function generator</td>
 <td>Trek</td>
 <td>615A-1 615-3</td>
 <td></td>
 </tr>
 <tr>
 <td>MVX10 microscope</td>
 <td>Olympus</td>
 <td></td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
 <tr>
 <td>QIClick digital CCD camera</td>
 <td>QImaging</td>
 <td>QIClick-F-CLR-12</td>
 <td>Optional (facilitates tracking of droplets on DMF hub)</td>
 </tr>
</tbody>
</table>

a versatile automated platform for micro-scale cell stimulation experiments

Étude des cellules en culture (analyse in vitro) a fourni des informations importantes sur les systèmes biologiques complexes. Méthodes et équipements conventionnels pour analyse in vitro sont bien adaptés à l&#39;étude d&#39;un grand nombre de cellules (≥ 10 5) des volumes millilitre échelle (≥ 0,1 ml). Cependant, il ya de nombreux cas dans lesquels il est nécessaire ou souhaitable de réduire la taille de la culture pour réduire la consommation des cellules d&#39;intérêt et / ou de réactifs nécessaires à leur culture, stimulation, ou de transformation. Malheureusement, les approches conventionnelles ne supportent pas une manipulation précise et reproductible des cultures de micro-échelle, et les systèmes automatisés microfluidique à base actuellement disponibles sont trop complexes et spécialisés pour une utilisation de routine par la plupart des laboratoires. Pour résoudre ce problème, nous avons développé une plate-forme technologique simple et polyvalent pour la culture automatisé, la stimulation et la récupération de petites populations de cellules (100 - 2.000 cellules) en volume à micro-échelles (1 - 20 pi). La plate-forme se compose d&#39;un ensemble de microcapillaires revêtues de fibronectine («chambres de perfusion de cellules&quot;), dans lequel les cultures de micro-échelle sont établis, maintenus et encouragés; un appareil numérique microfluidique (DMF) équipé de «transfert» microcapillaires (&quot;hub central &quot;), dont les cellules itinéraires et des réactifs vers et à partir des chambres de perfusion; une pompe à seringue à haute précision, qui transportent des puissances de matériaux entre les chambres de perfusion et le moyeu central, et une interface électronique qui permet de contrôler le transport de matières, ce qui est coordonné et automatisé via des scripts préétablis. À titre d&#39;exemple, nous avons utilisé la plate-forme pour faciliter l&#39;étude des réactions suscitées transcription dans les cellules immunitaires lors de défi avec des bactéries. L&#39;utilisation de la plate-forme nous a permis de réduire la consommation de cellules et de réactifs, de minimiser la variabilité expérience à l&#39;expérience, et de réorienter les mains sur le travail. Compte tenu des avantages qu&#39;il confère, ainsi que son accessibilité et sa polyvalence, our plate-forme doit trouver une utilisation dans une grande variété de laboratoires et d&#39;applications, et se révéler particulièrement utile pour faciliter l&#39;analyse des cellules et des stimuli qui sont disponibles en quantités limitées.

Comme une démonstration de la plate-forme automatisée, nous l&#39;avons utilisé pour réaliser une étude dans laquelle de petites populations de cellules immunitaires ont été cultivées dans des cultures de micro-échelle, contesté par des bactéries, et lysées pour l&#39;analyse hors plate-forme de réponses pro-inflammatoires (Figure 2 ). Chacune des six chambres de perfusion de cellules a été ensemencé avec 10 4 cellules immunitaires (macrophages mur...

Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Nous avons développé une culture de cellules automatisé et plate-forme d&#39;interrogation pour les expériences de stimulation cellulaire micro-échelle. La plate-forme offre un contrôle simple, polyvalent et précis dans la culture et la stimulation de petites populations de cellules, et la récupération de lysats pour les analyses moléculaires. La plate-forme est bien adapté aux études qui utilisent des cellules précieux et / ou des réactifs.

Une plate-forme automatisée polyvalent pour micro-échelle expériences de stimulation cellulaire

Study of cells in culture (in vitro analysis) has provided important insight into complex biological systems. Conventional methods and equipment for in ...

a-versatile-automated-platform-for-micro-scale-cell-stimulation

Research

JoVE Journal

Bioengineering

10.5K vues.  Sandia National Laboratories. Nous avons développé une culture de cellules automatisé et plate-forme d'interrogation pour les expériences de stimulation cellulaire micro-échelle. La plate-forme offre un contrôle simple, polyvalent et précis dans la culture et la stimulation de petites populations de cellules, et la récupération de lysats pour les analyses moléculaires. La plate-forme est bien adapté aux études qui utilisent des cellules précieux et / ou des réactifs.

Vidéo: A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments

Organotypic Collagen I Assay: A Malleable Platform to Assess Cell Behaviour in a 3-Dimensional Context

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and metastasis of tumor cells. Migration has been studied on glass coverslips in order to make cellular dynamics amenable to investigation by light microscopy. However, it has become clear that many aspects of cell migration depend on features of the local environment including its elasticity, protein composition, and pore size, which are not faithfully represented by rigid two dimensional substrates such as glass and plastic 1. Furthermore, interaction with other cell types, including stromal fibroblasts 2 and immune cells 3, has been shown to play a critical role in promoting the invasion of cancer cells. Investigation at the molecular level has increasingly shown that molecular dynamics, including response to drug treatment, of identical cells are significantly different when compared in vitro and in vivo 4. 
Ideally, it would be best to study cell migration in its naturally occurring context in living organisms, however this is not always possible. Intermediate tissue culture systems, such as cell derived matrix, matrigel, organotypic culture (described here) tissue explants, organoids, and xenografts, are therefore important experimental intermediates. These systems approximate certain aspects of an in vivo environment but are more amenable to experimental manipulation such as use of stably transfected cell lines, drug treatment regimes, long term and high-resolution imaging. Such intermediate systems are especially useful as proving grounds to validate probes and establish parameters required to image the dynamic response of cells and fluorescent reporters prior to undertaking imaging in vivo 5. As such, they can serve an important role in reducing the need for experiments on living animals.

A method is described for the preparation of a 3-dimensional matrix consisting of collagen type I and primary human fibroblasts. This organotypic gel serves as a useful substrate to assess invasive cell migration because it mimics basic features of tissue stroma and is amenable to many forms of microscopy.

Organotypiques de collagène I Dosage: Une plate-forme malléable pour évaluer le comportement des cellules dans un contexte 3-Dimensional

Cell migration is fundamental to many aspects of biology, including development, wound healing, the cellular responses of the immune system, and ...

Recherche

Bio-ingénierie

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.
GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of &tilde;10 pure enzymes requires less than 100 &mu;g of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).
Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.
All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (&beta;-glucosidase), EX3 (endo-&beta;1,4-xylanase, GH10), and BX (&beta;-xylosidase).
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GENPLAT (GLBRC Enzyme Platform) is an automated platform for discovery and optimization of enzyme cocktails for biomass degradation. It can be adapted to multiple feedstocks and mixtures of enzymes containing multiple components.

GENPLAT: une plate-forme automatisée pour Discovery enzymatique de la biomasse et l&#39;optimisation Cocktail

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This technology has shown potential in addressing previously challenging applications; including, delivery to primary immune cells, cell reprogramming, carbon nanotube, and quantum dot delivery. This vector-free microfluidic platform relies on mechanical disruption of the cell membrane to facilitate cytosolic delivery of the target material. Herein, we describe the detailed method of use for these microfluidic devices including, device assembly, cell preparation, and system operation. This delivery approach requires a brief optimization of device type and operating conditions for previously unreported applications. The provided instructions are generalizable to most cell types and delivery materials as this system does not require specialized buffers or chemical modification/conjugation steps. This work also provides recommendations on how to improve device performance and trouble-shoot potential issues related to clogging, low delivery efficiencies, and cell viability.

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This protocol provides detailed steps on how to use the system for a broad range of applications.

Presser cellule comme, microfluidique Delivery Platform intracellulaire robuste

Rapid mechanical deformation of cells has emerged as a promising, vector-free method for intracellular delivery of macromolecules and nanomaterials. This ...

A Novel Stretching Platform for Applications in Cell and Tissue Mechanobiology

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have contributed dramatically to the understanding of basic mechanobiology. These techniques have been extensively used to demonstrate how the onset and progression of various diseases are heavily influenced by mechanical cues. This article presents a multi-functional biaxial stretching (BAXS) platform that can either mechanically stimulate single cells or quantify the mechanical stiffness of tissues. The BAXS platform consists of four voice coil motors that can be controlled independently. Single cells can be cultured on a flexible substrate that can be attached to the motors allowing one to expose the cells to complex, dynamic, and spatially varying strain fields. Conversely, by incorporating a force load cell, one can also quantify the mechanical properties of primary tissues as they are exposed to deformation cycles. In both cases, a proper set of clamps must be designed and mounted to the BAXS platform motors in order to firmly hold the flexible substrate or the tissue of interest. The BAXS platform can be mounted on an inverted microscope to perform simultaneous transmitted light and/or fluorescence imaging to examine the structural or biochemical response of the sample during stretching experiments. This article provides experimental details of the design and usage of the BAXS platform and presents results for single cell and whole tissue studies. The BAXS platform was used to measure the deformation of nuclei in single mouse myoblast cells in response to substrate strain and to measure the stiffness of isolated mouse aortas. The BAXS platform is a versatile tool that can be combined with various optical microscopies in order to provide novel mechanobiological insights at the sub-cellular, cellular and whole tissue levels.

We present in this article a novel stretching platform that can be used to investigate single cell responses to complex anisotropic biaxial mechanical deformation and quantify the mechanical properties of biological tissues.

Une nouvelle plate-forme d&#39;étirement pour Applications dans cellulaire et tissulaire mécanobiologie

Tools that allow the application of mechanical forces to cells and tissues or that can quantify the mechanical properties of biological tissues have ...

A Microfluidic Platform for High-throughput Single-cell Isolation and Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet high-throughput, method to perform single-cell culture experiments. Here, we report a microfluidic chip-based strategy for high-efficiency single-cell isolation (~77%) and demonstrate its capability of performing long-term single-cell culture (up to 7 d) and cellular heterogeneity analysis using clonogenic assay. These applications were demonstrated with KT98 mouse neural stem cells, and A549 and MDA-MB-435 human cancer cells. High single-cell isolation efficiency and long-term culture capability are achieved by using different sizes of microwells on the top and bottom of the microfluidic channel. The small microwell array is designed for precisely isolating single-cells, and the large microwell array is used for single-cell clonal culture in the microfluidic chip. This microfluidic platform constitutes an attractive approach for single-cell culture applications, due to its flexibility of adjustable cell culture spaces for different culture strategies, without decreasing isolation efficiency.

Here, we present a protocol for isolating and culturing single cells with a microfluidic platform, which utilizes a new microwell design concept to allow for high-efficiency single cell isolation and long-term clonal culture.

Une plate-forme microfluidique pour l&#39;isolement à haut débit unique cellule et Culture

Studying the heterogeneity of single cells is crucial for many biological questions, but is technically difficult. Thus, there is a need for a simple, yet ...

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

Layered alginate Constructs: Une plate-forme pour la coopération en culture des populations de cellules hétérogènes

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

A High-throughput Cell Microarray Platform for Correlative Analysis of Cell Differentiation and Traction Forces

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly controlled, high-throughput engineered system for measuring the impact of arrayed biochemical signals on cell differentiation. Recent efforts using cell microarrays have demonstrated their utility for combinatorial studies in which many microenvironmental factors are presented in parallel. However, these efforts have focused primarily on investigating the effects of biochemical cues on cell responses. Here, we present a cell microarray platform with tunable material properties for evaluating both cell differentiation by immunofluorescence and biomechanical cell&#8211;substrate interactions by traction force microscopy. To do so, we have developed two different formats utilizing polyacrylamide hydrogels of varying Young&#39;s modulus fabricated on either microscope slides or glass-bottom Petri dishes. We provide best practices and troubleshooting for the fabrication of microarrays on these hydrogel substrates, the subsequent cell culture on microarrays, and the acquisition of data. This platform is well-suited for use in investigations of biological processes for which both biochemical (e.g., extracellular matrix composition) and biophysical (e.g., substrate stiffness) cues may play significant, intersecting roles.

Cell differentiation is regulated by a host of microenvironmental factors, including both matrix composition and substrate material properties. We describe here a technique utilizing cell microarrays in conjunction with traction force microscopy to evaluate both cell differentiation and biomechanical cell&#8211;substrate interactions as a function of microenvironmental context.

Un haut-débit Plate-forme Cell biopuces pour corrélative Analyse de la différenciation des cellules et des forces de traction

Microfabricated cellular microarrays, which consist of contact-printed combinations of biomolecules on an elastic hydrogel surface, provide a tightly ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Une méthode polyvalente de patterning Les protéines et les cellules

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

DNA Nanotubes as a Versatile Tool to Study Semiflexible Polymers

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of flexible polymers nor of rigid rods. Underlying filaments remain outstretched due to their non-vanishing backbone stiffness, which is quantified via the persistence length (lp), but they are also subject to strong thermal fluctuations. Their finite bending stiffness leads to unique, non-trivial collective mechanics of bulk networks, enabling the formation of stable scaffolds at low volume fractions while providing large mesh sizes. This underlying principle is prevalent in nature (e.g., in cells or tissues), minimizing the high molecular content and thereby facilitating diffusive or active transport. Due to their biological implications and potential technological applications in biocompatible hydrogels, semiflexible polymers have been subject to considerable study. However, comprehensible investigations remained challenging since they relied on natural polymers, such as actin filaments, which are not freely tunable. Despite these limitations and due to the lack of synthetic, mechanically tunable, and semiflexible polymers, actin filaments were established as the common model system. A major limitation is that the central quantity lp cannot be freely tuned to study its impact on macroscopic bulk structures. This limitation was resolved by employing structurally programmable DNA nanotubes, enabling controlled alteration of the filament stiffness. They are formed through tile-based designs, where a discrete set of partially complementary strands hybridize in a ring structure with a discrete circumference. These rings feature sticky ends, enabling the effective polymerization into filaments several microns in length, and display similar polymerization kinetics as natural biopolymers. Due to their programmable mechanics, these tubes are versatile, novel tools to study the impact of lp on the single-molecule as well as the bulk scale. In contrast to actin filaments, they remain stable over weeks, without notable degeneration, and their handling is comparably straightforward.

Semiflexible polymers display unique mechanical properties that are extensively applied by living systems. However, systematic studies on biopolymers are limited since properties such as polymer rigidity are inaccessible. This manuscript describes how this limitation is circumvented by programmable DNA nanotubes, enabling experimental studies on the impact of filament rigidity.

Nanotubes d’ADN comme un outil polyvalent pour l’étude des polymères semi-flexibles

Mechanical properties of complex, polymer-based soft matter, such as cells or biopolymer networks, can be understood in neither the classical frame of ...

A Multilayer Microfluidic Platform for the Conduction of Prolonged Cell-Free Gene Expression

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic regulatory circuits. The analysis of synthetic genetic regulatory networks in vivo is time consuming and suffers from a lack of environmental control, with exogenous synthetic components interacting with host processes resulting in undesired behavior. To overcome these issues, cell-free characterization of novel circuitry is becoming more prevalent. In vitro transcription and translation (IVTT) mixtures allow the regulation of the experimental environment and can be optimized for each unique system. The protocols presented here detail the fabrication of a multilayer microfluidic device that can be utilized to sustain IVTT reactions for prolonged durations. In contrast to batch reactions, where resources are depleted over time and (by-) products accumulate, the use of microfluidic devices allows the replenishment of resources as well as the removal of reaction products. In this manner, the cellular environment is emulated by maintaining an out-of-equilibrium environment in which the dynamic behavior of gene circuits can be investigated over extended periods of time. To fully exploit the multilayer microfluidic device, hardware and software have been integrated to automate the IVTT reactions. By combining IVTT reactions with the microfluidic platform presented here, it becomes possible to comprehensively analyze complex network behaviors, furthering our understanding of the mechanisms that regulate cellular processes.

The fabrication process of a PDMS-based, multilayer, microfluidic device that allows in vitro transcription and translation (IVTT) reactions to be performed over prolonged periods is described. Furthermore, a comprehensive overview of the hardware and software required to automate and maintain these reactions for prolonged durations is provided.

Une plate-forme microfluidique multicouche pour la conduite d'une expression génique prolongée sans cellules

The limitations of cell-based synthetic biology are becoming increasingly apparent as researchers aim to develop larger and more complex synthetic genetic ...