Name: a novel feeder-free system for mass production of murine natural killer cells in vitro
Uploaded: 2018-01-09T14:00:00
Description: Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

Denne undersøgelse blev støttet af den forskning tilskud Rådet af Hong Kong (GRF 468513, CRF-CUHK3-12R) og Innovation og teknologi fond i Hong Kong (ITS/227/15, ITS InP/164/16, ITS-InP/242/16), direkte tilskud til forskning-CUHK (2016.035) og Hong Kong Scholar Program.
H.-Y.L. udviklet og overvåget alle eksperimenter og bidraget til manuskriptet forberedelse. P.M.-K.T. udførte eksperimenter, analyseres data og bidraget til manuskriptet forberedelse. P.C.-T. T., J.Y.-F.C., J.S.-C., H., Q.-M.W., og G.-Y.L. indsamlet animalske prøver og deltog i dyreforsøg. J.S., X.-R.H., og K.-F.T. bidraget til manuskriptet forberedelse.

Protokollen er for at opnå og differentiere knoglemarv-afledte NK celler (BM-NK) baseret på tidligere udgivne metoder20,21,22. Alle procedurer med mus er blevet godkendt af dyr etik eksperimentelle udvalg (AEEC) ved den kinesiske University of Hong Kong.
1. forberedelse af OP9 betingede Medium
<ol><li>Kultur murine stroma cellelinje OP9 i alpha-MEM indeholdende 20% FBS, 100 U/mL penicillin G og 10...

<ol>
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I det foreliggende arbejde, har vi beskrevet en ny metode til fremstilling af knoglemarv-afledte murine NK celler in vitro. Celle feeder i det oprindelige system21,22 erstattes med succes af den betingede medium af OP9 celler, som i høj grad øget differentiering systemets stabilitet. Derudover systemet kan producere høj mængde og renhed af modne NK celler til in vitro- samt in vivo assays, som kan lette Mekanismestudier samt Transla...

Kræft progression er stort set afhængig af tumor mikromiljø1,2, herunder vært-afledte immunocytes, fx, NK-celler. Flere undersøgelser viste, at intratumoral NK celler er negativt korreleret med tumor progression3,4. Derudover viste kliniske undersøgelser, at NK adoptiv celleterapi er en mulig strategi for kræft5,6,7,8,9. NK cellebaserede cancer immunterapi foreslog for nylig som en terapeutisk mulighed for solide tumorer, men udfordringer findes som følge af udskillelsen af immunosuppressive cytokiner og downregulation for at aktivere ligander i mikromiljø af solide tumorer 10,11. Omdanne vækst faktor-β (TGF-β) er blevet foreslået til at spille en forebyggende rolle i carcinogenese, men paradoksalt nok kræftceller producerer også TGF-B1 til støtte for tumor udvikling12,13,14 , 15. TGF-β signalering kan undertrykke cytolytisk aktiviteten af NK celler via ned-regulering interferon lydhørhed og CD16-medieret interferon-gamma (IFN-γ) produktion in vitro-16,17, 18.
Selv om afbrydelse af TGF-β signalering i tumor mikromiljø kan være en mulig vej til at fjerne kræft, vil fuldstændig blokering af TGF-β signalering forårsage autoimmune sygdomme på grund af dets anti-inflammatoriske funktion, som det fremgår af udviklingen i bivirkninger herunder systemisk inflammation, modeller kardiovaskulære defekter og autoimmunitet i mus19. Dermed, forståelse arbejder mekanisme af TGF-β-medieret immunosuppression vil føre til identifikation af et tilgængeligt terapeutiske mål for behandling af kræft.
For at belyse de molekylære begivenheder nødvendige for NK celle udvikling, etableret Williams et al. en in vitro- system for differentiere murine knoglemarv hæmatopoietisk stamceller i NK celler20. Dette system letter i høj grad Mekanismestudier NK celle udvikling, herunder identifikation af roman stamfaderen til NK celler21. Men knoglemarv stamceller bør være kulturperler i systemet med støtte OP9 stromale celler som en feeder lag20,21, og denne heterogene cellen befolkningen i høj grad begrænser den videre anvendelse af gen-forstyrre værktøjer (fxsiRNA-medieret genhæmning) specifikt udlignet til de differentiering NK celler.
Her, beskriver vi en feeder-fri system, der er blevet udviklet af yderligere ændring i vitro system af Williams mfl20. I vores system, OP9 stromale feeder celler er ikke påkrævet, og i stedet OP9 betingede medium bruges uden at påvirke differentiering af NK celler in vitro-, og dette seneste føre os til at afdække, TGF-β er i stand til at fremme kræft progression via undertrykke E4bp4-afhængige NK celle udvikling i tumor mikromiljø22. Dette nye system med held giver en baggrund-fri metode til at belyse de molekylære mekanisme af NK celle udvikling under særlige betingelser (f.eks.høj TGF-B1, siRNA-medieret genhæmning, osv.) i vitro.

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	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:297px;">OP9 cell line</td>
			<td style="width:129px;">ATCC</td>
			<td style="width:344px;">ATCC&#174; CRL-2749&#8482;</td>
			<td style="width:343px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">MEM &#945;, no nucleosides</td>
			<td>Gibco</td>
			<td>22561021</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Fetal Bovine Serum</td>
			<td>Gibco</td>
			<td>10500064</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PBS, pH 7.4</td>
			<td>Gibco</td>
			<td>10010049</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-7</td>
			<td>PEPROTECH</td>
			<td>217-17</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine SCF</td>
			<td>PEPROTECH</td>
			<td>250-03</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine Flt3-Ligand</td>
			<td>PEPROTECH</td>
			<td>250-31L</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Recombinant Murine IL-2</td>
			<td>PEPROTECH</td>
			<td>212-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Lipofectamin RNAiMAX Transfection Reagent</td>
			<td>Invitrogen</td>
			<td>1377815</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">IC Fixation Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-8222-49</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Flow Cytometry Staining Buffer&#160;</td>
			<td>eBioscience</td>
			<td>00-4222-26</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">PE-conjugated anti-mouse CD244</td>
			<td>eBioscience</td>
			<td>12-2441-83</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Cy3-conjugated anti-mouse NKp46</td>
			<td>Bioss</td>
			<td>bs-2417R-cy3</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Nonsense control (NC)</td>
			<td>Ribobio</td>
			<td>siN05815122147</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">siRNA against mouse E4BP4 mRNA</td>
			<td>Ribobio</td>
			<td>N/A</td>
			<td>5&#8242;-GAUGAGGGUGUA 
			GUGGGCAAGUCUU-3&#8242;</td>
		</tr>
	</tbody>
</table>

a novel feeder-free system for mass production of murine natural killer cells in vitro

Natural killer (NK) celler tilhører den medfødte immunsystem og er en første-line anti-cancer immun forsvar; men de undertrykkes i tumor mikromiljø og den underliggende mekanisme er stadig stort set ukendt. Manglen på en konsekvent og pålidelig kilde af NK celler begrænser forskning fremskridt af NK celler immunitet. Her rapporterer vi en in vitro- system, der kan give høj kvalitet og mængde af knoglemarv-afledte murine NK celler under en feeder-fri tilstand. Endnu vigtigere, vise vi også, at siRNA-medieret genhæmning held hæmmer E4bp4-afhængige NK celle modning ved hjælp af dette system. Denne roman in vitro- NK celler differentiere system er således en biomateriale løsning for immunitet forskning.

Repræsentative resultater opnås efter den beskrevne protokol. Samlede knoglemarv suspension celler blev dyrket under ordningen feeder-fri differentiering i 11 dage; betydelig forøgelse af spredning sats blev observeret af dag 7 sammenlignet med antallet samlede celler på dag 0 (fig. 1A). Modne NK celler med høj nuklear til cytoplasmatisk ratio og granula-rige cytoplasma morfologi blev fundet af dag 6 i systemet (figu...

Watch this Scientific Journal Video about A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro at JoVE.com

A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

Vi præsenterer her, en protokol til at masseproducere genhæmning murine NK celler ved hjælp af en feeder-fri differentiering system for mekanistisk undersøgelse in vitro og in vivo.

En roman Feeder-fri ordning for masseproduktion af Murine naturlige dræberceller In Vitro

Natural killer (NK) cells belong to the innate immune system and are a first-line anti-cancer immune defense; however, they are suppressed in the tumor ...

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A Novel Feeder-free System for Mass Production of Murine Natural Killer Cells In Vitro

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In vitro Generation af murint Plasmacytoide dendritiske celler fra fælles Lymfoid progenitorer ved hjælp af AC-6 Feeder System

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The ability to rapidly secrete cytokines upon stimulation is a functional characteristic of the invariant natural killer T (iNKT) cell lineage. iNKT cells ...

Flow Cytometric Analysis of Natural Killer Cell Lytic Activity in Human Whole Blood

Flowcytometrisk analyse of Natural Killer Cell lytisk aktivitet i humant fuldblod

NK cell cytotoxicity is a widely used measure to determine the effect of outside intervention on NK cell function. However, the accuracy and ...

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