Name: a microfluidic technique to probe cell deformability
Uploaded: 2014-09-03T13:00:00
Description: Watch this Scientific Journal Video about A Microfluidic Technique to Probe Cell Deformability at JoVE.com

Forfatterne vil gerne anerkende Lloyd Ung til konstruktive bidrag i tidlige versioner af denne teknik, Dr. Jeremy Agresti for pres cap design tips, og Dr. Dongping Qi for hans hjælp i opdigte trykdækslet. Vi er taknemmelige for laboratorier M. Teitell og P. Gunaratne for at levere en bred vifte af celleprøver til test. Vi er taknemmelige for National Science Foundation (KARRIERE Award DBI-1.254.185), UCLA Jonsson Comprehensive Cancer Center og UCLA Clinical and Translational Science Institute for at støtte dette arbejde.

1. mikrofluidindretning Design BEMÆRK: Enheden design har fire grundlæggende funktionelle områder: indgangsport, celle filter, konstriktion array, og udgangsport (Figur 1). Det overordnede design kan anvendes til en bred vifte af celletyper, med mindre justeringer af dimensioner. Forudsat her er et par grundlæggende design anbefalinger sammen med enhedens parametre, der er effektive for et udvalg af både primære og udødeliggjorte celler. <ol><li> Vælg bredden af ko...

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Her giver vi et omfattende eksperimentelle procedure til analyse af deformation af celler i transit gennem trange mikrofluidkanaler anvendelse af tryk-drevet flow. En MATLAB script muliggør automatiseret databehandling (supplerende materiale); en opdateret version af koden opretholdes ( <a href="www.ibp.ucla.edu/research/rowat" target="_blank">www.ibp.ucla.edu/research/rowat</a> ). Mere generelt kan de teknikker, der præsenteres her, tilpasses i mange cellebaserede mikrofluide analyser, inklusive effekten af cytoskele...

Ændringer i celleform er kritiske i mange biologiske sammenhænge. For eksempel, erythrocytter og leukocytter deformere gennem kapillærer, der er mindre end deres egen diameter 1. I metastase skal kræftceller deformere gennem smalle interstitielle huller samt indviklet karsystem og lymfe netværk til frø på sekundære steder 2. Til at sondere den fysiske opførsel af de enkelte celler, præsentere mikrofluidenheder en ideel platform, der kan tilpasses til at studere en række celle adfærd, herunder deres evne til at vandre gennem smalle huller 3 og til passivt at deformere gennem micron skala forsnævringer 3- 9. Polydimethylsiloxan (PDMS) mikrofluidenheder er optisk transparent, så celle deformationer skal visualiseres ved hjælp af lys mikroskopi og analyseres ved hjælp af grundlæggende billedbehandling værktøj. Desuden kan arrays af forsnævringer være præcist defineret, så analyse af flere celler samtidig med etgennemløb, der overstiger mange eksisterende teknikker 10,11. Her præsenterer vi en detaljeret forsøgsprotokol til sondering celle deformerbarhed ved hjælp af &#39;Cell Deformer&#39; PDMS mikrofluidindretning. Enheden er designet således, at cellerne passage gennem sekventielle indsnævringer; denne geometri er almindelig i fysiologiske sammenhænge, ​​såsom pulmonal kapillærbane 12. At måle celle deformerbarhed, transit tid giver en bekvem metrik, der er let måles som den tid, der kræves for en individuel celle til transit gennem en enkelt indsnævring 4,6. For at opretholde et konstant trykfald over de trange kanaler under celle forsendelse, vi bruger trykdrevet strømning. Vores protokol indeholder detaljerede instruktioner om enheden design og fabrikation, enhedens drift efter trykdrevet strømning, forberedelse og billeddannelse af celler, samt billedbehandling til at måle tiden for celler at deformere gennem en række forsnævringer. Vi inkludererbåde enhedens design og forarbejdning vision data kode som supplerende filer. Som et repræsentativt udsnit af data, viser vi celle transittid gennem en serie af indsnævringer som en funktion af antallet af indsnævringer passeret. Analyse af tidsplanen for celler til transit selvom smalle forsnævringer af en mikrofluid enhed kan afsløre forskelle i deformerbarheden af en række celletyper 4,5,13. Enheden demonstreret her entydigt overvåger celle transit gennem en serie af micron skala forsnævringer; dette motiv emulerer snoet vej at celler opleve i omløb, og giver også mulighed for sondering yderligere fysiske egenskaber af celler, såsom afslapning tid.

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			<td height="42" style="height:42px;width:216px;">Name of Material/ Equipment</td>
			<td style="width:71px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pluronic F-127 Block Copolymer Surfactant&#160;</td>
			<td style="width:71px;">Fisher Scientific&#160;</td>
			<td style="width:119px;">8409400</td>
			<td style="width:167px;">Produced by BASF, also available through Sigma</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">PDMS base and crosslinker</td>
			<td style="width:71px;">Essex Brownell</td>
			<td style="width:119px;">DC-184-1.1</td>
			<td style="width:167px;">Product commonly named Sylgard 184 Elastomer</td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Oxygen plasma discharge unit</td>
			<td style="width:71px;">Enercon</td>
			<td style="width:119px;">Dyne-A-Mite 3D Treater</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Biopsy Punch, Harris Uni-Core (0.75 mm)</td>
			<td style="width:71px;">Ted Pella, Inc.</td>
			<td style="width:119px;">15072</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight Ferrule, 1/32&#34;</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-113</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Fingertight III Fitting, 10-32</td>
			<td style="width:71px;">Upchurch Scientific</td>
			<td style="width:119px;">UP-F-300X</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyetheretherketone (PEEK) tubing, outer diameter = 1/32&#34;or 0.79 mm</td>
			<td style="width:71px;">Valco</td>
			<td style="width:119px;">TPK.515-25M</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">polyethylene (PE-20) tubing, 0.043&#34; or 1.09 mm</td>
			<td style="width:71px;">Becton Dickinson</td>
			<td style="width:119px;">427406</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Pressure regulator</td>
			<td style="width:71px;">Airgas or Praxair</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Polyurethane tubing, 5/32&#8221; OD</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5648K284</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Push-to-connect fittings</td>
			<td style="width:71px;">McMaster Carr</td>
			<td style="width:119px;">5111K91</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="35">
			<td height="35" style="height:35px;width:216px;">Voltage to Pressure (E/P) Electropneumatic Converter</td>
			<td style="width:71px;">Omega</td>
			<td style="width:119px;">IP413-020</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">16-bit,250 kS/S, 80 Analog Inputs Multifunction DAQ</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">NI PCI 6225-779295-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Analog Connector Block-Screw Terminal</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SCB-68-776844-01</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">LabView System Design Software</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="52">
			<td height="52" style="height:52px;width:216px;">Matlab Software</td>
			<td style="width:71px;">The MathWorks, Inc.</td>
			<td style="width:119px;">Matlab R2012a</td>
			<td style="width:167px;">Code requires the Image Processing Toolbox</td>
		</tr>
		<tr height="53">
			<td height="53" style="height:53px;width:216px;">Shielded Cable</td>
			<td style="width:71px;">National Instruments</td>
			<td style="width:119px;">SHC68-68</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

a microfluidic technique to probe cell deformability

Her detaljeret udformning, fremstilling og anvendelse af en mikrofluidindretning at evaluere deformerbarhed af et stort antal individuelle celler på en effektiv måde. Typisk kan data for ~ 10 2 celler kan erhverves inden for en 1 time eksperiment. En automatiseret billedanalyse program muliggør effektiv post-eksperiment analyse af billeddata, så behandling for at være fuldstændig inden for et par timer. Vores apparat geometri er unik ved, at celler skal deformere gennem en række micron skala forsnævringer, hvorved den indledende deformation og tidsafhængig lempelse af individuelle celler, der skal analyseres. Anvendeligheden af ​​denne metode til human promyelocytisk leukæmi (HL-60) celler er påvist. Kørsel celler til at deformere gennem micron skala forsnævringer hjælp af tryk-drevet flow, observere vi, at den menneskelige promyelocytiske (HL-60) celler momentant okkludere første indsnævring til en median tid på 9,3 msek før passage hurtigere gennem den efterfølgende snøreioner med en median transittid på 4,0 msek pr konstriktion. Derimod all-trans-retinsyre-behandlede (neutrofil-type) HL-60-celler okkludere første indsnævring kun 4,3 msek før passage gennem de efterfølgende indsnævringer med en median transittid på 3,3 msek. Denne metode kan give indsigt i den viskoelastiske natur af celler, og i sidste ende afslører de molekylære oprindelsen af ​​denne adfærd.

At undersøge deformerbarheden forskellige celletyper, humane myeloide leukæmiceller (HL-60), differentierede neutrofile celler, mus lymfocytceller og human ovariekræft cellelinjer (OVCAR8, HEYA8) vurderes ved hjælp af &#39;Cell Deformer &quot;mikrofluid teknik. Repræsentative resultater for transittiden af HL-60-og neutrofil-type HL-60-celler viser tidsplanen for en enkelt celle til transit gennem en serie indsnævringer, som vist i figur 6. Transittiden måles for en population af individuelle cel...

Watch this Scientific Journal Video about A Microfluidic Technique to Probe Cell Deformability at JoVE.com

A Microfluidic Technique to Probe Cell Deformability

Vi demonstrere en mikrofluidik-assay til måling af tidshorisonten for celler til transit gennem en sekvens af mikron-skala forsnævringer.

En Mikrofluid Teknik til sonde Cell deformerbarhed

Here we detail the design, fabrication, and use of a microfluidic device to evaluate the deformability of a large number of individual cells in an ...

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Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

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 ...

Analyzing Mixing Inhomogeneity in a Microfluidic Device by Microscale Schlieren Technique

Analyserer Blanding Inhomogeniteter i en mikrofluidapparat af Individuel Schlieren Technique

In this paper, we introduce the use of microscale schlieren technique to measure mixing inhomogeneity in a microfluidic device. The microscale schlieren ...

Microfluidic Genipin Deposition Technique for Extended Culture of Micropatterned Vascular Muscular Thin Films

Microfluidic genipin Deposition Teknik for Extended Culture of mikromønstrede Vaskulære Muskuløse Thin Films

The chronic nature of vascular disease progression requires the development of experimental techniques that simulate physiologic and pathologic vascular ...

Using Adhesive Patterning to Construct 3D Paper Microfluidic Devices

Brug Adhesive mønstret Konstruér 3D Paper mikrofluidenheder

We demonstrate the use of patterned aerosol adhesives to construct both planar and nonplanar 3D paper microfluidic devices. By spraying an aerosol ...

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

En Mikrofluid platform for High-throughput Single-celle Isolering og Kultur

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 ...

Rapid Subtractive Patterning of Live Cell Layers with a Microfluidic Probe

Hurtig Subtraktiv mønster af levende cellelag med et Mikrofluid Probe

The microfluidic probe (MFP) facilitates performing local chemistry on biological substrates by confining nanoliter volumes of liquids. Using one ...

Multi-step Variable Height Photolithography for Valved Multilayer Microfluidic Devices

Multi-trins Variabel Højde Fotolitografi for med ventiler Multilayer mikrofluidenheder

Microfluidic systems have enabled powerful new approaches to high-throughput biochemical and biological analysis. However, there remains a barrier to ...