Name: cell squeezing as a robust, microfluidic intracellular delivery platform
Uploaded: 2013-11-07T19:45:00
Description: Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Vi takker T. Shatova for nyttige drøftelser om eksperimenterende design og dataanalyse. Den støtte og ekspertise G. Paradis, er personalet i flowcytometri kerne ved Koch Institut, og personalet i den Microsystems Technology Laboratory ved Massachusetts Institute of Technology taknemmeligt anerkendt. Dette arbejde blev støttet af National Institutes of Health Tilskud RC1 EB011187-02, DE013023, DE016516, EB000351, og delvist af National Cancer Institute Cancer Center Support (Core) Tilskud P30-CA14051 og MPP-09Call-Langer-60.

1.. Opbevaring <ol><li> Opbevar reservoirerne, holdere, O-ringe og mikrofluidenheder i 70% ethanol. Brug en beholder (f.eks krukke eller bægerglas), som har et låg for at forhindre fordampning og forurening med støv eller uden partikler. Placer enhederne i en beholder (1), reservoirer og O-ringe i en anden beholder (2), og indehavere i den tredje (3). </li></ol> Bemærk: Brugen af ​​70% ethanol til opbevaring er at opretholde sterilitet. Hvis de eneste dele af løsningen er etha...

<ol>
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Visse aspekter af den beskrevne eksperimentelle procedure (dvs. andre end chip design og driftshastighed faktorer), kan skal optimeres afhængig af celletype og levering materiale systemet påføres. Den diskussion, der følger adresser nogle af de mest almindelige faktorer at overveje, når designe eksperimenter. For at forbedre leveringen signalet for fluorescens-mærkede forbindelser, er man nødt til at tage fat på kilderne til baggrundsfluorescens. Surface binding og endocytos...

Levering af makromolekyler til cellen cytoplasma er et afgørende skridt i terapeutiske og forskningsmæssige applikationer. Nanopartikel medieret levering, for eksempel, har vist potentiale i genterapi 1,2, mens protein levering er en lovende middel til at påvirke cellulære funktion i både kliniske 3 og laboratorie 4 indstillinger. Andre materialer, såsom små molekyle narkotika, kvantepunkter eller guld nanopartikler, er af interesse i ansøgninger fra kræftmedicin 5,6 til intracellulær mærkning 7,8, og enkelt molekyle sporing 9. Cellemembranen er hovedsagelig impermeabel for makromolekyler. Mange eksisterende teknikker bruger polymere nanopartikler 10,11, liposomer 12, eller kemiske modifikationer 13 for at lette membran afbrydelse eller endocytotisk levering. I disse metoder, levering effektivitet og celle levedygtighed er ofte afhængige af strukturen i the målmolekyle og celletype. Disse metoder kan være effektive ved levering af strukturelt ensartede materialer, såsom nukleinsyrer, men er ofte dårligt egnet til levering af mere strukturelt forskellige materialer, såsom proteiner 14,15 og nanomaterialer 7. Desuden endosomet forstyrrelser mekanisme, at de fleste af disse metoder er afhængige af, er ​​ofte ineffektiv og dermed efterlader meget materiale fanget i vesikel strukturer 16. Endelig mener metoder, der ofte er udviklet til brug med etablerede cellelinjer ikke oversætte godt til primære celler. Membran perforeringselementer fremgangsmåder, såsom elektroporering 17,18 og sonoporation 19, er et attraktivt alternativ i visse anvendelser, men de er kendt for at forårsage levedygtighed lav celle og kan begrænses ved ladningen af mållevering materiale. Hurtig mekanisk deformation af celler, en mikrofluid tilgang til levering, har for nylig demonstrated sine fordele i forhold til de nuværende teknikker i forbindelse med celle omprogrammering 20 og nanomateriale levering 21. Denne metode er baseret på mekanisk sprængning o cellemembranen at lette cytosolisk levering af materiale til stede i den omgivende buffer. Systemet har vist så potentiale i tidligere udfordrende celletyper (f.eks primære immunceller og stamceller) og materialer (fx antistoffer og kulstof nanorør). Heri er generelle fremgangsmåde til at anvende disse anordninger til intracellulær levering af target makromolekyler beskrevet. Proceduren er generaliserbare til de fleste celletyper og levering materialer, anbefales det dog, at man foretager en kortfattet optimering af betingelser, som nærmere beskrevet i vores tidligere rapporteret design guidelines 20, for eventuelle tidligere urapporteret applikationer. Til dato, er systemet blevet anvendt med succes til levering af RNA, DNA, guld nanopartikler, kvantepunkter, kulstof-nanorør, proteins, og dextran polymerer 20,21.

<table border="0" cellpadding="0" cellspacing="0" width="777">
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	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:375px;">Device Holder &#38; Plastic reservoir</td>
			<td style="width:136px;">SQZ Biotechnologies</td>
			<td style="width:149px;">Holder</td>
			<td style="width:117px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LSRFortessa Analyzer</td>
			<td>Becton Dickinson</td>
			<td>N/A</td>
			<td>Flow cytometry machine used at the Koch Institute Core Facilities</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Microfluidic device</td>
			<td>SQZ Biotechnologies</td>
			<td>Cell Squeeze</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">O-Rings</td>
			<td>McMaster</td>
			<td>9452K311</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Pressure system to operate device</td>
			<td>SQZ Biotechnologies</td>
			<td>Pressure System</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Tweezers</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Ultrasound bath</td>
			<td></td>
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</table>

cell squeezing as a robust, microfluidic intracellular delivery platform

Hurtig mekanisk deformation af celler har vist sig som et lovende, vektor-fri metode til intracellulær levering af makromolekyler og nanomaterialer. Denne teknologi har vist potentiale i håndteringen tidligere udfordrende applikationer, herunder levering til primære immunceller, celle omprogrammering, kulstof nanorør og kvantepunktet levering. Denne vektor-fri microfluidic platform afhængig mekanisk sprængning af cellemembranen til at lette cytosolisk levering af målmaterialet. Heri beskriver vi den detaljerede metode til brug for disse mikrofluidenheder herunder enhed forsamling, cellepræparation og systemdrift. Denne levering tilgang kræver en kort optimering af enhedens type og driftsbetingelser for tidligere urapporteret applikationer. De medfølgende vejledning er generaliserbare til de fleste celletyper og levering materialer da dette system ikke kræver specialiserede buffere eller kemisk modifikation / konjugeringsreaktioner trin. Dette arbejde giver ogsås anbefalinger om hvordan man kan forbedre enhedens ydeevne og problemfri skyde potentielle problemer relateret til tilstopning, lav levering effektivitetsgevinster og cellernes levedygtighed.

Figur 1 indeholder en beskrivende skematisk mikrofluid delivery system. Figurerne 2a-b illustrerer typiske resultater fra behandling af HeLa-celler med forskellige apparatkonstruktioner i nærvær af fluorescens-konjugeret 3 kDa dextran 20. Hvis proceduren er fulgt korrekt, vil systemets ydeevne være følsom over for enhedstype og driftshastighed. Derfor bør man optimere disse forhold til en given anvendelse, før man går videre til mere komplekse eksperimenter. I rækken ...

Watch this Scientific Journal Video about Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform at JoVE.com

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Hurtig mekanisk deformation af celler har vist sig som et lovende, vektor-fri metode til intracellulær levering af makromolekyler og nanomaterialer. Denne protokol indeholder detaljerede trin om, hvordan man bruger systemet til en bred vifte af applikationer.

Cell Klemme som en robust, Mikrofluid Intracellulær Delivery Platform

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

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