A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device.

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by  Adewola, Adeola F.

JournalofVisualizedExperiments www.jove.com
VideoArticle
A Multi-Parametric Islet Perifusion System within a Microfluidic Perifusion Device
AdeolaF. Adewola1,Yong Wang1,Tricia Harvat1,DavidT. Eddington2,Dongyoung Lee1,Jose Oberholzer1,2
1DepartmentofSurgery,
2DepartmentofBioengineering,
Correspondenceto:[email protected]
URL:http://www.jove.com/index/Details.stp?ID=1649
DOI:10.3791/1649
Citation:Adewola A.F.,Wang Y.,Harvat T.,Eddington D.T.,Lee D.,Oberholzer J.(2010).AMulti-ParametricIsletPerifusionSystemwithinaMicrofluidicPerifusionDevice.JoVE.35.
http://www.jove.com/index/Details.stp?ID=1649,doi:10.3791/1649
Abstract
Amicrofluidicisletperifusiondevicewasdevelopedfortheassessmentofdynamicinsulinsecretionofmultipleisletsandsimultaneous
fluorescenceimagingofcalciuminfluxandmitochondrialpotentialchanges.Thedeviceconsistsofthreelayers:firstlayercontainsanarrayof
microscalewells(500μmdiameterand150μmdepth)thathelptoimmobilizetheisletswhileexposedtoflowandmaximizetheexposedsurface
areaoftheislets;thesecondlayercontainsacircularperifusionchamber(3mmdeep,7mmdiameter);andthethirdlayercontainsaninlet-mixing
channelthatfansoutbeforeinjectionintotheperifusionchamber(2mminwidth,19mminlength,and500μminheight)foroptimizingthemixing
efficiencypriortoenteringtheperifusionchamber.Thecreationofvariousglucosegradientsincludingalinear,bellshape,andsquareshapesalso
canbecreatedinthemicrofluidicperifusionnetworkandisdemonstrated.
Protocol
A. Microfabrication of 3-layer microfluidic perifusion device
BottomWellMasterProtocol(150μmdeepwells)
1. Cleanthewaferusingarazorbladeifneeded.CleanwithAcetone,Methanol,andIPA.Performplasmatreatmentat50Wattsfor30s.
2. SpinSU8-100@2000rpm.[Step1:500rpm,10s,100rpm/s,Step2:2000rpm,30s,300rpm/s].
[NOTE:donotholdthewaferwithtweezersafterspinningSU8]
3. Softbakethewaferat65°Cfor20minandat95°Cfor50min.
4. ThewaferisexposedtoUVlightthroughadesiredmask.Dosefor150μmheightis650mJ/cm2.
5. Postexposurebakethewaferat65°Cfor1minandat95°Cfor12min.
6. DevelopthewaferinSU8developerfor15min.
MicrochannelMasterProtocol(500μmdeepwells)
1. Cleanthewaferusingarazorbladeifneeded.CleanwithAcetone,Methanol,andIPA.Performplasmatreatmentat50Wattsfor30s.
2. SpinSU8-2150@1000rpm.[Step1:500rpm,10s,100rpm/s,Step2:1000rpm,30s,300rpm/s].
3. Softbakethewaferat65°Cfor15minandat95°Cfor2hrsand30min.
4. ThewaferisexposedtoUVlightthroughadesiredmask.Exposuredosefor650μmheightis685mJ/cm2.
5. Postexposurebakethewaferat65°Cfor5minandat95°Cfor35min.
6. DevelopthewaferinSU8developerfor20-30min.
PDMSsolutionpreparation
1. Polydimethylsiloxane(PDMS)solutionispreparedbythoroughlymixing10partsofsiliconeelastomerwith1partofcuringagentofa
standardSylgard184kit.
2. ThebubblesgeneratedinthePDMSsolutionduringthemixingprocessareremovedusingavacuumdesiccator.
3. Thebubble-freePDMSsolutionisslowlydispensedontotheSU8mastersandanemptyPetridishforthethirdlayer.
4. Thetemperatureofthehotplateissetto75°CandthePDMSiscuredatthistemperaturefor2hrs
5. Inlets,outletsandexchangewellsarepunchedoutusingtheappropriatesizeholepuncher.
6. Thelayersarebondedtogetheronaglassslide(size0.1mm)usingahandheldplasmadevice.
B. Experimental setup
1. Flow70%ethanolthroughthemicro-devicetosterilize.FlowDIwatertowashouttheethanol.Perfuse50mlof0.5%BSAthroughthe
devicetopreventnonspecificadsorptionofinsulintothemicrochannelwalls.
2. TheglucoserampgradientandotherrelatedgradientsgeneratedbyLabViewsoftwarethatcommunicateswiththesyringepumpsaretested
tomakesurethegradientsarestable.
3. 25-30miceisletswereincubatedwith5μMFura-2/AM(acalciumindicator,Molecularprobes,CA)and2.5μMRhodamine123(Rh123,a
mitochondrialpotentialsindicator,Sigma,MO)for30minat37°CinKrebs-Ringerbuffer(KRB)containing2mMglucose
4. Themiceisletsarecarefullypipettedintomicrofluidicperifusiondevicethroughtheinletport.
5. ThemicrofluidicnetworkisthensetupbyconnectingtheinlettothesyringepumpsusingTygontubingandaY-connectorandtheoutletto
fractioncollector.Thedevicesitsonaheatingstage(37°C)onthemicroscopeandtheinlettubingisheatedonahotplatetokeepthe
temperatureofthechamberandsolutionat37°C.
6. Immediatelyaftersetup,themiceisletsareperifusedwithKRBcontaining2mMglucosefor10minandthenaglucoseramp(2mM25mM)
for25min.
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7. Time-lapseimagesarecollectedandanalyzedevery15sbySimplePCIsoftware.Theperifusateisalsocollectedeveryminuteusinga
fractioncollectortoanalyzetheinsulinsecretionusingELISAkit.
Representative Results
Mouseisletswereperifusedwithalineargradientof2-25mMglucose.AsshowninFigure1,calciuminfluxandinsulinsecretionaretriggered
afterabout13minutesofperifusion,correspondingto6mMglucose.ChangesinMitochondrialpotentialsareseenearlierasexpected,atabout
11minutes.Thisdatademonstratestheadvantageofusingthismicrofluidicnetworktocharacterizeisletphysiology.
Figure1.Physiologicalresponsestoisletcellstimulation.
Discussion
Traditionalisletperifusionsystems(macroscaleandmicroscale)havesomelimitationsincludingcomplexofsetupanddesign,hightechnical
requirements,anddifficultytocreateuser-prescribedchemicalgradientsinthesystem.Themicrofluidicperifusionsystemdescribedhere
overcomestheselimitationswithsimplegeometryofdesignandfabrication.Moreimportant,thissystemcanbeintegratedwithfluorescence
imagingapproachthatprovideasauniquetooltostudyisletphysiology.Thesystemdemonstratedwithhighsignal-noiseratioand
spatial-temporalresolutionofthesefluorescencesignals.Thecurrentprototypedevicealsocanholdmultipleperifusionsetupsinonechipand
providedifferenttypesofmicroenvironmentsforwidespreadapplicationpurposes.
Acknowledgements
ThisworkwassupportedbyAAUWinternationalfellowshiptoAdeolaAdewola,NIH/NCRR(U42RR023245)toJoseOberholzer,andTheChicago
DiabetesProject.
References
1. Mohammed,JS.,Wang,Y.,Harvat,TA.,Oberholzer,J.,Eddington,DT.Microfluidicdeviceformultimodalcharacterizationofpancreaticislets.
LabChip9,97-106(2009).
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