Name: a versatile automated platform for micro-scale cell stimulation experiments
Uploaded: 2013-08-06T14:00:00
Description: Watch this Scientific Journal Video about A Versatile Automated Platform for Micro-scale Cell Stimulation Experiments at JoVE.com

Forfatterne takker Ronald F. Renzi og Michael S. Bartsch for deres bidrag til udformning og udvikling af DMF enheder og DMF hub. Denne forskning var fuldt støttet af Laboratory Directed Forskning og Udvikling programmet på Sandia National Laboratories. Sandia er en multi-program laboratorium ledes og drives af Sandia Corporation, et helejet datterselskab af Lockheed Martin Corporation, for det amerikanske Department of Energy National Nuclear Security Administration under kontrakt DE-AC04-94AL85000.

Denne generelle protokol er designet til at understøtte anvendelsen af platformen til en bred vifte af undersøgelser aspekter specifikke for repræsentativ undersøgelse beskrevet i denne rapport er adskilt i parentes Figur 2 illustrerer en repræsentativ undersøgelse udført ved hjælp af protokollen.. Bemærk, at der i protokollen, er alle &quot;instruere&quot; kommandoer automatiseret ved brug af forudbestemte scripts. Bemærk også, at trin 2 kan udføres parallelt med trin 1 (fx under t...

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Vi har udviklet en enkel og alsidig automatiseret platform for mikroskala cellekultur og stimulering eksperimenter. Platformen gør det muligt for os at arbejde med små mængder kultur og celle populationer (1 - 20 ul og 100 - 2.000 celler per kammer), kultur størrelser kunne yderligere reduceret ved brug af microcapillaries af mindre diameter. Arbejde på disse skalaer reducerer omkostningerne til rutinemæssige undersøgelser og gør gennemførlige undersøgelser, der kræver brug af ædle reagenser og / el...

Studiet af celler opretholdt i kultur (in vitro analyse) har givet et uvurderligt indblik i de grundlæggende principper og molekylære mekanismer, der gælder komplekse biologiske systemer og menneskers sundhed. De traditionelle metoder til kultur, stimulation, og indsamling af celler til analyse, der udnytter petriskåle og mikrotiterplader, var designet til undersøgelse af store populationer af celler (≥ 10 5) i milliliter-skala kulturvolumener (≥ 0,1 ml). Men der er mange tilfælde, hvor der kun begrænsede mængder af celler er tilgængelige (f.eks primære celler), eller små populationer af celler er ønskelige (fx at reducere celle-til-celle variation i befolkningen), eller krævede reagenser er vanskelige at opnå eller uoverkommeligt dyrt (f.eks rensede celle-secernerede faktorer). Sådanne spørgsmål kan med held behandles af nedtrapning kultur størrelse, som har den ekstra fordel at reducere forbruget af allereagenser kræves for in vitro-analyse 1,2. Desværre konventionelt udstyr og metoder understøtter ikke præcis og reproducerbar manipulation af mikro-skala kulturer og MicroFluidics-baserede automatiske systemer øjeblikket er til rådighed 3-11 er for komplekse og specialiserede til rutinemæssig brug af de fleste laboratorier. I denne rapport beskriver vi montering og brug af en enkel og alsidig teknologiplatform til automatiseret kultur, stimulation, og inddrivelse af små populationer af celler (100 - 2.000 celler) i mikro-skala mængder (1 - 20 ul). Platformen arkitektur (figur 1) er modulopbygget: et sæt af fibronectin-belagt microcapillaries (&quot;celle perfusion kamre&quot; module) tjener som stedet for etablering, vedligeholdelse og stimulering af mikro-skala kulturer og en digital microfluidics (DMF ) 12,13 enhed udstyret med &quot;overførsel&quot; microcapillaries (&quot;central hub&quot;-modulet) 14,15 ruter cellerog reagenser til og fra perfusion kamre. DMF giver brugeren mulighed for individuelt at tage flere dråber samtidigt og til at ændre eller genbestille manipulationer (dvs. omkonfigurere prøve forarbejdning tog) uden at ændre enhedens hardware. Dens enorme fleksibilitet er tydelig i sin nye rolle som en nøgleteknologi i en bred vifte af applikationer, herunder cellekultur 16,17, enzymassays 18,19, immunobestemmelser 20,21, DNA-analyse 22,23, protein behandling, 24,25 og klinisk prøvebehandling. 26,27 Vores centrale hub drager fordel af fleksibiliteten til DMF-enheder, og yderligere forbedrer det ved tilsætning af mikrokapillær grænseflader, som giver mulighed for at foretage en delmængde af manipulationer (f.eks cellekultur) i specialiserede perifere moduler, snarere end på DMF enheden selv. Opdeling af forarbejdning tog på denne måde forenkler også design af plaTForm arkitektur (ikke nødvendigt at bygge en DMF enhed, der kan udføre alle procestrin) og letter dets udvikling som nye funktioner er påkrævet (simpelthen integrere nye perifere moduler som nødvendigt). Transport af celler og reagenser inden for det centrale knudepunkt drives af electrowetting kræfter, der genereres ved sekventiel aktivering af elektroder i DMF-enhed 13,28, transport til, fra og inden for de perfusion kamre er drevet af trykændringer genereret af en høj præcision sprøjte pumpen. Alle disse flydende bevægelser styres via en enkel elektronisk grænseflade og automatiseret ved brug af forudbestemte scripts. Som et repræsentativt eksempel, viser vi brug af platformen for studiet af transkriptionelle responser fremkaldt hos immunceller ved udfordring med bakterier (figur 2). Udføre disse eksperimenter på platformen muligt for os at arbejde med et lille antal celler (~ 1.000 pr eksperital betingelse), minimerer eksperiment-til-eksperimentet variabilitet spare reagenser, og re-direct hands-on arbejdskraft. I betragtning af de fordele, det giver, samt dets tilgængelighed og alsidighed, bør denne platform finde brug i en bred vifte af laboratorier og applikationer og bevise især nyttig i at lette analysen af ​​celler og stimuli, der er tilgængelige i begrænsede mængder.

<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

Undersøgelse af celler i kultur (in vitro analyse) har givet vigtig indsigt i komplekse biologiske systemer. Konventionelle metoder og udstyr til in vitro-analysen er velegnede til at studere af et stort antal celler (≥ 10 5) i milliliter-skala volumener (≥ 0,1 ml). Men der er mange tilfælde, hvor det er nødvendigt eller ønskeligt at nedskalere kultur størrelse at reducere forbruget af cellerne af interesse og / eller reagenser, der er nødvendige for deres kultur, stimulation eller forarbejdning. Desværre har konventionelle metoder understøtter ikke præcis og reproducerbar manipulation af mikro-skala kulturer og MicroFluidics-baserede automatiske systemer øjeblikket er til rådighed, er for kompleks og specialiseret til rutinemæssig brug af de fleste laboratorier. For at løse dette problem, har vi udviklet en enkel og alsidig teknologiplatform til automatiseret kultur, stimulation, og inddrivelse af små populationer af celler (100 - 2.000 celler) i mikro-skala volumens (1 - 20 ul). Platformen består af et sæt af fibronectin-belagt microcapillaries (&quot;celle perfusion kamre&quot;), inden for hvilke mikro-skala kulturer er etableret, vedligeholdes og stimuleres, en digital microfluidics (DMF)-enhed udstyret med &quot;overførsel&quot; microcapillaries (&quot;central hub &quot;), som ruter celler og reagenser til og fra perfusion kamre, et høj præcision sprøjte pumpe, hvilke beføjelser transport af materialer mellem perfusion kamre og den centrale hub og en elektronisk grænseflade, som giver kontrol over transport af materialer, som er koordineres og automatiseret via forudbestemte scripts. Som et eksempel brugte vi platformen til at lette undersøgelse af transskriptionelle responser fremkaldt i immunceller ved udfordring med bakterier. Brug af platformen muligt for os at reducere forbruget af celler og reagenser, minimerer eksperiment-til-eksperimentet variabilitet, og re-direkte hands-on arbejdskraft. I betragtning af de fordele, det giver, samt dets tilgængelighed og alsidighed, our platform skulle finde anvendelse i en bred vifte af laboratorier og applikationer, og bevise især nyttig i at lette analysen af ​​celler og stimuli, der er tilgængelige i begrænsede mængder.

Som en demonstration af den automatiske platform, brugte vi det til at gennemføre en undersøgelse, hvor små populationer af immunceller blev dyrket i mikro-skala kulturer udfordres med bakterier, og lyseret for off-platform analyse af pro-inflammatoriske responser (figur 2 ). Hver af seks celle perfusion kamre blev podet med 10 4 immunceller (P388D.1 murine makrofager) resuspenderes i 10 pi vækstmedium. Efter en tilslutning periode (37 ° C i 2 timer) og et me...

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

Vi har udviklet en automatiseret cellekultur og forhør platform for mikro-skala cellestimulering eksperimenter. Platformen tilbyder enkel, alsidig og præcis kontrol i at dyrke og stimulere små populationer af celler, og inddrive lysater til molekylær analyser. Platformen er velegnet til undersøgelser, der anvender værdifulde celler og / eller reagenser.

En alsidig Automatiseret platform for Micro-skala cellestimulering Eksperimenter

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

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