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 ønsker å takke Lloyd Ung for konstruktive innspill i tidlige versjoner av denne teknikken, Dr. Jeremy Agresti for trykklokket design tips, og Dr. Ping Qi for hans hjelp i fabrikasjon trykklokket. Vi er takknemlige for laboratorier av M. Teitell og P. Gunaratne for å gi en rekke celleprøver for testing. Vi er takknemlige for National Science Foundation (KARRIERE Award DBI-1254185), UCLA Jonsson Comprehensive Cancer Center, og UCLA Clinical og Translasjonell Science Institute for å støtte dette arbeidet.

1. microfluidic Device Design MERK: Enheten designen har fire grunnleggende funksjonelle regioner: entry port, cellefilter, innsnevring array, og exit port (figur 1). Den generelle designen kan brukes til et bredt spekter av celletyper, med mindre justeringer av dimensjoner. Forutsatt her er noen grunnleggende design anbefalinger sammen med enhetsparametere som er effektive for et utvalg av både primære og udødeliggjort celler. <ol><li> Velg bredden innsnevring array-ka...

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Her gir vi en omfattende eksperimentell prosedyre for å analysere deformasjon av celler i transitt gjennom snørt microfluidic kanaler ved hjelp av trykkraft. En MATLAB script muliggjør automatisert databehandling (Supplemental Material); en oppdatert versjon av koden er opprettholdt ( <a href="www.ibp.ucla.edu/research/rowat" target="_blank">www.ibp.ucla.edu/research/rowat</a> ). I videre forstand kan de teknikker som er presentert her tilpasses i mange cellebaserte assays microfluidic, herunder virkningen av cytoske...

Endringer i celleform er avgjørende i en rekke biologiske sammenhenger. For eksempel, erytrocytter og leukocytter deformeres gjennom kapillærer som er mindre enn sin egen diameter 1. I metastasering, må kreftceller deformert gjennom trange interstitiell hull samt kronglete blodkar og lymfekar nettverk til frø på sekundære områder to. Å sondere den fysiske oppførselen til enkeltceller, presentere microfluidic enheter en ideell plattform som kan tilpasses til å studere en rekke celle atferd inkludert deres evne til å migrere gjennom smale hull 3 og passivt deformeres gjennom mikron-skala constrictions 3- ni. Polydimethylsiloxane (PDMS) microfluidic enheter er optisk transparent, slik at celle deformasjoner å bli visualisert ved hjelp av lysmikroskopi og analysert ved hjelp av grunnleggende bildebehandlingsverktøy. Videre kan sammensetninger av innsnevringer bli nøyaktig definert, slik at analyse av flere celler samtidig med engjennomstrømming som overgår mange eksisterende teknikker 10,11. Her presenterer vi en detaljert forsøksprotokoll for sondering celle formbarhet ved hjelp av &quot;Cell Deformer &#39;PDMS microfluidic enhet. Anordningen er utformet slik at cellene passasje gjennom sekvensielle innsnevringer; denne geometri er vanlig i fysiologiske sammenhenger, som for eksempel lunge-kapillære bed 12. For å måle celle deformerbarhet, gir transittid en enkel beregning som er lett måles som den tid som kreves for en enkelt celle til transitt gjennom en enkelt innsnevring 4,6. For å opprettholde et konstant trykkfall over de innsnevrede kanaler under celle transitt, bruker vi trykkraft. Vår protokoll omfatter detaljerte instruksjoner om enheten konstruksjon og fabrikasjon, betjeningen av trykkraft, preparering og avbildning av celler, så vel som bildebehandling for å måle tid for cellene til å deformeres gjennom en rekke innsnevringer. Vi inkludererbåde enhets design og visjon databehandling kode som supplerende filer. Som et representativt utvalg av data, viser vi celletransittiden gjennom en rekke innsnevringer som en funksjon av antall innsnevringer passaged. Analyse av tidsskalaen for celler til transitt skjønt trange innsnevringer av en mikrofluidteknisk enhet kan vise forskjeller i deformasjon av en rekke celletyper 4,5,13. Anordningen vist her entydig undersøkelser celle transitt gjennom en serie av mikron-skala innsnevringer; dette designet emulerer kronglete sti som cellene opplever i omløp og også gjør sondering flere fysiske egenskapene til cellene som avslapping 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 har vi detalj utforming, fabrikasjon og bruk av en mikrofluid enhet for å evaluere deformerbarhet av et stort antall enkeltceller på en effektiv måte. Vanligvis kan data for ~ 10 2 celler kan erverves i løpet av en 1-timers eksperimentet. En automatisert bildeanalyse-program gjør det mulig effektiv etter eksperiment analyse av bildedata, slik prosessering for å være fullstendig i løpet av få timer. Vår geometri enheten er unik ved at cellene må deformeres gjennom en serie av mikron-skala innsnevringer, og dermed muliggjør den innledende deformasjon og tidsavhengig avslapning av individuelle celler som skal undersøkes. Anvendeligheten av denne metode til human promyelocytiske leukemi (HL-60)-celler er vist. Kjøring celler til å deformere gjennom mikron-skala restriksjonar ved hjelp trykkraft, observerer vi at menneskelig Promyelocytic (HL-60) celler øyeblikk occlude den første innsnevring for en median tid på 9,3 msek før passering raskere gjennom den påfølgende constrictioner med en median overføringstiden av 4,0 ms pr innsnevring. I motsetning til dette, all-trans retinsyre-behandlet (nøytrofil-type) HL-60-celler som okkluderer den første innsnevring i bare 4,3 ms før passering gjennom de etterfølgende innsnevringer med en midlere transittid på 3,3 msek. Denne metoden kan gi innsikt i viskoelastisk natur celler, og til slutt avdekke molekylære opprinnelsen til denne atferden.

For å undersøke formbarhet av ulike celletyper, menneske myelogen leukemi celler (HL-60), differensierte nøytrofile celler, mus lymfocyttceller og menneskelige eggstokkreft cellelinjer (OVCAR8, HEYA8) er evaluert ved hjelp av &quot;Cell Deformer &#39;microfluidic teknikk. Representative resultater for overføringstiden av HL-60-og nøytrofil-type HL-60-celler som viser tidsskalaen for en enkelt celle til transitt gjennom en rekke innsnevringer, som vist i figur 6. Transittiden måles for en populasjo...

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

A Microfluidic Technique to Probe Cell Deformability

Vi demonstrerer en MicroFluidics-baserte analysen for å måle tidsskalaen for celler til transitt gjennom en sekvens av mikron-skala restriksjonar.

En mikrofluid Technique å sondere Cell Fleksibilitet

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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Design of a Cyclic Pressure Bioreactor for the Ex Vivo Study of Aortic Heart Valves

The aortic valve, located between the left ventricle and the aorta, allows for unidirectional blood flow, preventing backflow into the ventricle. Aortic ...

Cell Squeezing as a Robust, Microfluidic Intracellular Delivery Platform

Cell klemme som en robust, Microfluidic Intracellulær Levering 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

Analysere Blanding inhomogeneity i et mikrofluid Device av mikro 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 Nedfall Technique for Extended Culture of Micropatterned Vaskulær 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

Ved hjelp av lim Mønstring å konstruere 3D Paper microfluidic enheter

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 plattform for High-throughput encellede 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

Rapid Subtraktiv Mønstring av levende cellelag med en 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-trinns variabel Høyde Fotolitografi for Valved Multilayer microfluidic enheter

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

Large-area Scanning Probe Nanolithography Facilitated by Automated Alignment and Its Application to Substrate Fabrication for Cell Culture Studies

Stor-området skanning sonde Nanolithography tilrettelagt av automatiserte justering og dens anvendelse til underlaget fabrikasjon for cellen kulturstudier