The first method is published. It should be noted that in this published part of the project, we left out the idea of virtual networks. This topic will be covered in section 4.4.
4.3 Citation and original contribution
Robert Samhaber∗, Manuel Schottdorf∗, Ahmed El Hady∗, Kai Br¨oking, Andreas Daus, Chris-tiane Thielemann, Walter St¨uhmer, and Fred Wolf: “Growing neuronal islands on multi-electrode arrays using an accurate positioning-µCP device”, J Neurosci Methods 257(1): 194-203 (2016) [∗ eq. contribution]418
I assembled the construction sheets and generated the overview figure (Fig. 1); I measured the reproducibility of the patterning procedure by quantifying the PLL deposition on each stamping site (Fig. 3) and I produced patterned cultures and performed the electrophysiological measure-ments (Fig. 6). In addition, I designed the website for the supplemental online information, wrote the text together with all authors and I am corresponding author.
ContentslistsavailableatScienceDirect
Journal of Neuroscience Methods
j ou rn a l h o m epa g e : w w w . e l s e v i e r . c o m / l o c a t e / j n e u m e t h
Basicneuroscience
Growing neuronal islands on multi-electrode arrays using an accurate positioning- CP device
RobertSamhabera,b,d,e,f,1,ManuelSchottdorfa,b,d,e,∗,1,AhmedElHadya,b,d,e,f,1, KaiBrökinga,b,d,e,AndreasDausc,ChristianeThielemannc,WalterStühmera,e, FredWolfb,d,e,f,g,∗
aMax-Planck-InstituteforExperimentalMedicine,Dept.MolecularBiologyofNeuronalSignals,Hermann-Rein-Str.3,37075Göttingen,Germany
bMaxPlanckInstituteforDynamicsandSelf-Organization,Dept.NonlinearDynamics,AmFaßberg17,37077Göttingen,Germany
cFacultyofEngineering,UniversityofAppliedScience,WürzburgerStraße45,63743Aschaffenburg,Germany
dBernsteinCenterforComputationalNeuroscience,Göttingen,Germany
eBernsteinFocusNeurotechnology,Göttingen,Germany
fSFB-889CellularMechanismsofSensoryProcessing,Göttingen,Germany
gFacultyofPhysics,Georg-August-UniversitätGöttingen,Göttingen,Germany
h i g h l i g h t s
•AprecisionpatterningprotocolforhippocampalneuronsonMultielectrodearrays(MEAs)usingsimpleandwidelyavailableequipment.
•Ascalableprototypeforaversatilemechanicalpatternaligner.
•Increasedyieldofelectricalactivityrecordinginpatternedneuronalculture.
a rt i c l e i n f o
Background:Multi-electrodearrays(MEAs)allownon-invasivemulti-unitrecordingin-vitrofrom cul-tured neuronal networks. For sufficient neuronal growth and adhesion on such MEAs, substrate preparationisrequired.PlatingofdissociatedneuronsonauniformlypreparedMEA’ssurfaceresults intheformationofspatiallyextendedrandomnetworkswithsubstantialinter-samplevariability.Such culturesarenotoptimallysuitedtostudytherelationshipbetweendefinedstructureanddynamicsin neuronalnetworks.Toovercometheseshortcomings,neuronscanbeculturedwithpre-defined topol-ogybyspatiallystructuredsurfacemodification.SpatiallystructuringaMEAsurfaceaccuratelyand reproduciblywiththeequipmentofatypicalcell-culturelaboratoryischallenging.
Newmethod:Inthispaper,wepresentanovelapproachutilizingmicro-contactprinting(CP)combined withacustom-madedevicetoaccuratelypositionpatternsonMEAswithhighprecision.Wecallthis techniqueAP-CP(accuratepositioningmicro-contactprinting).
Comparisonwithexistingmethods:OtherapproachespresentedintheliteratureusingCPforpatterning eitherreliedonfacilitiesortechniquesnotreadilyavailableinastandardcellculturelaboratory,orthey didnotspecifymeansofprecisepatternpositioning.
Conclusion:Herewepresentarelativelysimpledeviceforreproducibleandprecisepatterningina stan-dardcell-culturelaboratorysetting.ThepatternedneuronalislandsonMEAsprovideabasisforhigh throughputelectrophysiologytostudythedynamicsofsingleneuronsandneuronalnetworks.
©2015ElsevierB.V.Allrightsreserved.
∗Correspondingauthorsat:TheoreticalNeurophysicsResearchGroup,NonLinearDynamicsDepartment,MaxPlanckInstituteforDynamicsandSelf-Organization,Am Fassberg17,37077Göttingen,Germany.Tel.:+4905515176423;fax:+4905515176409.
E-mailaddresses:manuel@nld.ds.mpg.de(M.Schottdorf),fred@nld.ds.mpg.de(F.Wolf).
1 Theseauthorscontributedequallytothiswork.
Theculturing andsurvivaloflivingcellsinvitrorequiresthe preparation of suitable conditions in an artificial environment.
In particular,in orderto studytheelectrophysiological proper-tiesofdissociatedneurons,aclosecontactbetweenthecellsand therecordingelectrodeshastobeestablished.Thenegative sur-facechargeandhydrophobicnatureofunmodifiedglasssurfaces areknowntocounteractattachmentandgrowthofneurons.One waytomodifythesurface propertiesina favourablewayisby coatingwithgrowthandadhesionpromotingmoleculestoallow attachment,developmentandcellsurvival.Multi-electrodearrays (MEAs)offeraversatileandwell-establishedtoolforboth, non-invasivelystudyingactivitypatternsinneuronal networksona widerangeofspatialscales(Grossetal.,1977,1995,1997;Stett etal., 2003;Hofmannand Bading,2006; Hofmannet al.,2011;
Schottdorfetal.,2012)andbiosensorapplications(Keeferetal., 2001;Chiappaloneetal.,2003;Selingeretal.,2004;Martinoiaetal., 2005;Xiangetal.,2007).
MEAsaredevicesinwhichathinlayerofaconducting mate-rialintheformofanelectrodearrayisembeddedontothesurface ofaglasssubstrate,allowingfornon-invasiveparallelrecording andstimulationofelectricalactivityatmultiplesitesfromacell culture.Platingdissociatedneuronsonauniformlycoatedculture substrateresultsinrandomnetworkformationthatarehighly vari-ableintheirdetailedtopology.Awaytoalignthegrowthofcell processeswiththepredefinedtopologyoftheMEAandtoreduce inter-culturevariabilityistoapplypatternedsubstrate modifica-tionsthat arealignedwiththepredefinedtopologyof theMEA layout.
CulturingneuronsonMEAsforextracellularstimulationand sig-nalrecording,togetherwiththeabilitytopreciselyandreliably pattern neuronal networks, is thus a crucial step in the devel-opmentofneuroelectronichybridssuchasbiosensors,neuronal prosthesesandneuroelectroniccircuits.
Patterningneuronsinapredefinedtopologyrequiresthat geo-metricparameterslikepatternlayout,dimensionandalignment toasubstratecanbeadjustedreproduciblyandprecisely.Several methodstoachieveapredefinedtopologyinculturedneuronal net-workshavebeenproposedinthepast:topographical-patterning andchemical-patterning.Pioneeredinthe1960s,different topo-graphical patterning techniques included etching groves on a substrate and lithographic procedures to directly model three dimensionalfeaturesonaculturesubstrate(NiemeyerandMirkin, 2004).Chemicalpatterningmethodsincludepatterneddeposition ofadhesionpromotingproteins(Wheeleretal.,1999;Scholletal., 2000;Nametal.,2004a,b).Usingthecellrepellingpropertiesof polyethylenglycol(PEG)throughaphoto-lithographicprocesswas alsoshowntoeffectivelydirectcellgrowth(KumarandWhitesides, 1994).Allofthesemethods,however,requirespecialised equip-mentnot readilyavailable in astandardcell-culture laboratory setting.Additionally,thealignmentofthepatterntobecultured witha givensubstratelayout requiresadditionalhigh-precision equipment.
More recently, seminal work on patterning using carbon nanotubeshasbeenperformedbythegroupofYaelHanein (Shein-Idelson et al., 2011) where islands of carbon nanotubes were deposited onthe electrodes leading to thepreferential growth ofclustersofneuronsovertheelectrodes.One-dimensional neu-ronalcultures,whichprovideaplatformtostudythepropagation speedofneuronal signals,havebeenrealizedonmultielectrode arrayusingacombinationofadhesiveandproteinrepelling coat-ing(JacobiandMoses,2007).Glialislandsonwhichmonolayers
siloxan (PDMS) micro stamps allows for a relatively simple approach to transfer adhesion promoting molecules such as Lamininorpoly-d-lysinetosubstratesurfacesathighspatial res-olution(Wheeleretal.,1999;Laueretal.,2001a,b;Jamesetal., 2004;Changetal.,2006;Junetal.,2007).Whilethismethoddoes requireaphoto-lithographylaboratoryinordertoproducea posi-tivetemplateforthestamps,continuedaccessisnotrequiredonce thestamphasbeenproduced.
Inthecurrentmethodpaperwepresentasimplepositioning deviceandmicro-contactprintingtechnique(accuratepositioning micro-contactprinting,AP-CP).
TocharacterizetheutilityoftheAP-CPprocedure,wedesigned anisland-patternfitting theelectrodelayoutof aMEA.Circular patternswereestablishedbymicro-contactprintingtheadhesion promoterontheMEA.Theislandswerealignedtotheelectrodes, allowingfor thegrowthof isolatedpopulationsof neurons.We show thattheAP-CPtechnique yieldsreproducible and topo-logically defined neuronal islands arbitrarily aligned with the electrodesofaMEA.Wealsopatternedneuronsinaone dimen-sional geometry allowing electrophysiological measurement of activitypropagationthroughaonedimensionalneuronalculture.
Ourstudythuspresentsasimple,preciseandreliablepatterning techniquethatcanserveasanelementarybutcrucialcomponent forthroughputelectrophysiologyofsingleneuronsandneuronal networks.
2. Materialsandmethods
2.1. Masterproductionthroughphoto-lithography
Accordingtothedesignedpattern,achromecoatedsoda-lime maskwasproducedbyelectronbeamlithography(ML&C,Jena).
Photoresist layers (AZ9260;Microchemicals, Ulm,Germany) of 20mwerespin-coated onglasswafersandsubsequently soft-bakedat100◦Cfor12min.Structuringofthephotoresist-layerwas obtainedbyexposuretoUVirradiationfor12mininclosecontact withthemaskcarryingthenegativepattern.Withpropercareand handling,nowearonthemasters couldbeobservedduringthe courseofthisstudy.Inthisstudy,weusedthreepredesigned pat-terns,twowhichrealized“Islands”patternsof64islandsofeither 90mor60mdiametereachseparatedby200m(Fig.2A).The secondpatternwasdesignedasa“Highway”pattern(Fig.6A)of 100minwidthand2mminlength(Fig.6A).
2.2. PDMSpreparation
PDMSpreparationwasperformedbymixingthePDMSSilicone Elastomer Baseand Curing Agent(Sylgard® 184; DowCorning, WiesbadenGermany)inaproportionof10:1.Theprepared vol-umewasstirredvigorouslybyhandand subsequentlydegassed underavacuumbelljar:Thelidlessvolumecontainingthemixed PDMSagentswasplacedinthebelljarandevacuatedwitha vac-uumpump.After3min,thepumpwasturnedoffandthebelljar leftinlowpressure(100–300hPa)for20mintoallowallair bub-blestoescape.ThePDMSexhibitseffervescenceduringevacuation.
Slowerapplicationoflowpressurepreventedthis.
2.3. Themould
ThestampswerecastfromPDMSintoastainlesssteelmould, theinteriorsurfacesofwhich hadbeenturnedtoasmooth
fin-Fig.1. Mechanicalandopticalsetupofthestampingdevice.Thedegreesoffreedomofthedevicearedistributedasfollows:(A)Thestampcanbemovedinz-direction,whereas theMEA(Multi-electrodearray)canbetranslatedinthexy-plane,androtatedaroundtheC-axis(B)Opticalsetup:Thetransparentstampisilluminatedfromabove.The positioningofthestampcanbecontrolledbymeansofawebcam,ontowhichtheMEAisimagedbymeansofanachromaticlens.(C)Photographofthestampingdevice.
(D)Thestamp,asseenfrombelowthroughaMEAbythewebcam.Forclarity,adropofblueinkhasbeenplacedontheelectrodes.Thestamppatternisinevencontactover allsites(E)Micrographofthestampsandthemould.
filledtoitsupperedgewithPDMSandthethreadedholding fix-turepressedintotheliquidpolymer(Fig.1E).Brushingtheinside ofthemouldwithasmallamountof10%SDSfacilitatedremoving thestampsafterthePDMSwasfullycured.Themastersbearing thePDMSfilledmouldswerekeptatroomtemperaturefor48hto allowthemixturetopolymerize.Fastercuringathigher temper-aturesresultedindistortionofthepattern(Wuetal.2003).The curedstampswereremovedfromthemouldsandstoredindouble distilledwateruntilneeded.
2.4. Mechanicalsetup
faceof analuminiumholdermouldedintothestamp.Thisface pressesagainstthecontactsurfacemachinedontotheplunger. Pro-videdtheabutmentiskeptclean,thesurfaceofthestampscanbe keptnormaltotheaxisoftheplungerwithinseveralsecondsof arc.Therefore,itisunnecessarytoproviderotationaldegrees of freedomaroundtheA,andBaxesinthestampingmechanismfor adjustment.
Thestampingpress,however,mustprovideadjustmentsforthe remainingfourdegreesoffreedom:First,theMEAmustberotated aroundtheC-axis,sothattheelectrodefieldcanbebroughtinto thesameorientationasthegridofmicrostamps.Then,theMEA
pattern.Thismeansthattheendpositionofthestamphastobe adjustedtoanaccuracyof±10m.Otherwise,thepatternwillbe depressedsofarthatthemicrostampsundergoanelastic deforma-tion,similartoEulerianbuckling,whichresultsininstantaneous contactbetweentheMEAandthestampsubstrate.Tothisend,a stoppingscrewofsuitablyfinepitchwasusedtoregulatethefinal positionofthestamprelativetotheMEAsurface.Themechanical setupthusconsistsofamicrometriccross-table,allowingfor posi-tioningoftheMEAwithinafewminthex–y-plain.Thisstageis mountedonarotarystagewithfinemovement,providingarotary degreeoffreedomalongtheCaxis.
The stampis fixed to anotherlinear stage with cross-roller bearings,allowingforprecisemovementinthez-axis.Thestamp consistsofaclearblockofSylgard184,castontoathreaded stamp-holder,thebackofwhichisthreadedtoallowfixingittothestamp plunge.Thebackofthisholderalsohasanaccuratelocatingface matchingasimilarfaceontheplunger.Thestamppistonisactuated byaneccentricactingagainstaroller-bearing,fixedtotheslideofa verticalstage.Theplungerisheldinadovetailfixedtothisslide.It isbeingheldtherebyasmallmagnetandcanbeclampedbymeans ofasmallscrew.Thisarrangementallowsforsomelimitedtravel betweentheslideandtheactualplunger,atthesametime ensur-ingthattheorientationoftheplungeriskeptasverticalaspossible.
Alladjustmentscanbemonitoredbyaninvertedmicroscopebuilt intotheapparatus,consistingofanachromat(f=18.5mm,f/1.5) belowtheMEA,andawebcamfixedtothesideofthemechanism (seeFig.1B).Sincethestampmaterialistransparent,thestamp pistonandthealuminium havebeenbored outandanLEDhas beenfixedintothebodyoftheplunger,providingenoughlightfor thecontactofthestampwiththeMEAsurfacetobeeasily recog-nized(Fig.1D).Acomputerrenderingofthestampingmachineis includedalongwiththeexactdesignofallpartsofthesetupand alistofcomponentsonthefollowingwebsite:http://www.nld.ds.
mpg.de/∼manuel/website/index.html.
Thecostofthesetupissmallintermsofmaterialsusedasmany oftheelementscanbeharvestedfromavailableoldmachinesor components.Here,weusedacheapcommercialwebcamforthe invertedmicroscopeand theC-axismanipulatorhasbeentaken fromamicroscopystage.Itisimportanttonotethatthespecific choiceof the manipulators,the webcam or the dichroic is not importantas longastheothermachine componentsare modi-fiedaccordingly.Publishingtheconstructionsheetsalongwiththis papershouldallowadjustingoursetupaccordingtothematerials available.
2.5. Substrate&stampsurfacepreparation
SterileMEAs(60MEA200/30iR-Ti;MultiChannelSystems, Reut-lingen,Germany)wereincubatedinpurifiedfoetalcalfserum(FCS;
Gibco)for30minandwashedoncewithdoubledistilledwaterand lefttodry.Asolutionof1%3-glycidoxypropyltrimethoxysilane (3-GPSNametal.,2004a,b;SigmaAldrich,Taufkirchen,Germany)in ToluenewasaddedtotheculturechamberoftheMEAfor20min andsubsequentlywashedthreetimeswithToluene.MEAswere thenbakedat100◦Cfor1handlefttocooldowntoroom temper-atureunderalaminarflowhoodpriortostamping.Subsequently thepreparedPDMSstampsweretakenoutofthedoubledistilled waterandanyremainingwaterwasremovedbysuction.Stamps weresterilizedbydippingin70%EtOHfor10s.ExcessEtOHwas removedbysuctionandstampswerelefttodryfor5min.
Adropoftheanionicdetergentsodiumdodecylsulphate(SDS;
10%w/v;SigmaAldrich-Aldrich,Taufkirchen,Germany)wasadded
ity ofthePDMS stamp(Changet al.,2003).The SDSwasdried under a nitrogen stream, washed with double distilled water and driedwithnitrogenagain. Aliquotsofa 0.1mg/mlsolution of poly-l-lysine conjugated with the fluorescent label fluores-ceinisothiocyanat(PLL-FITC)(70,000–150,000MW;SigmaAldrich, Taufkirchen,Germany)inphosphate-bufferedsaline(pH7.4)were thawed for 1h at37◦C in a water bath and vigorouslyshaken byhandevery15mintodissolveclustersofcoagulatedPLL-FITC (Wuetal.2003).50ldropletsofthesolutionwereaddedtothe patterned sideof thestamps andincubatedin thedarkfor1h.
Excessliquid wasremovedand thestampswerelefttodry for 10mintoallowall moisturetoevaporate.It wascriticaltothe stampingprocessnottoallowthePLL-FITCdroplettoevaporate beforeremovingitbysuction.Thestainlesssteelmouldscontaining the stampswerethen placedinto theplungerofthe mechani-calstampingdevice,alignedwiththeMEAandstampeduntothe substrate.
2.6. Cellculture
CellcultureswerepreparedaccordingtoBreweretal.(1993).
Hippocampal neurons were obtained from Wisteria WU rat embryosat18daysofgestation(E18).Thepregnantratwas anaes-thetizedbyCO2.Theembryoswereremovedbyacaesareansection, decapitatedandtransferredtocooledpetridishes.Theskullcavity wasopenedandthebrainremoved.Hippocampiweresurgically extractedandtransferredtoaHEPES(Invitrogen,Germany)buffer.
The supernatant was removed and the extracted hippocampi were trypsinized in a Trypsin/Ethylenediaminetetraacetic acid (EDTA)(trypsin:0.05%;EDTA:0.02%;SigmaAldrich,Taufkirchen, Germany)bufferfor15minat37◦C.Trypsinizedcellswerethen transferred to a 10% Foetal calf serum (FCS) solution. Thor-ough trituration using a syringe and a needle with a diameter of 1mm followed. Thecell suspensionwas thencentrifuged at 1200rpmfor2min.Thepelletwasre-suspendedin2mlof serum-freeB27/Neurobasal(B27:5%;Gibco)mediumsupplementedwith 0.5mMglutamineandBasicFibroblastGrowthFactor(bFGF).Cells were countedwitha Neubauerimprovedcounting chamber. A dropletof∼100lcellsuspensioncontaining50,000cells/mlwas addedontopoftheelectrodefieldoftheMEAs.Thisdensitywas chosentopreventtheformationofneuronalcellclusters,as dete-riorationofthepatternwasobservedathigherdensities.Lower densitiesledtolowersurvivabilityofneuronsaftermorethan7 daysinvitro.TheMEAswerethenkeptinanincubatorprovidinga humidifiedatmospherecontaining5%CO2at37◦Cfor4htoallow thecellstosettle.1mloftheB27/Neurobasalmediumwasthen addedtothecellchamber.Mediumwaschangedeverysevendays.
Allanimalswerekeptandbredintheanimalhouseofthe Max-Plank-InstituteofExperimentalMedicineaccordingtoEuropean andGermanguidelinesforexperimentalanimals.Animal exper-iments were carried out with authorization of the responsible federalstateauthority.
2.7. Immunocytochemistry
Patterned cultures on MEAs were used for immunocyto-chemistry after 14 days in vitro. Cultures were fixed with 4%
paraformaldehydeinphosphatebuffer(pH7.4)for3minat4◦C and subsequently washed three timeswithphosphate-buffered saline(PBS).Unspecificbindingsiteswereblockedbyincubation with3%AlbuminPBSfor30minatroomtemperaturebeforecells werepermeabilizedwithTritonX-100(0.5%inPBS,5min,4◦C).
Pri-Table1
Percentageofpopulatedislandsonthe60-islandpattern.
No.populatedislands (%)
Percentageofpopulatedislandsoftheislandpatternconsistingof60islandsaligned toastandardMEAlayout(60electrodes)after7daysinvitro(n=5;mean=60%;
standarddeviation=±3.9%).
thenappliedovernightat4◦C.AfterrinsingwithPBS,secondary antibodiesfromdonkey wereappliedfor2hwithadilution of 1:1000(alexa647anti-mouseIgG,alexa488anti-rabbit(Abcam)).
MEAsweresealedwithroundcoverslips(15mmdiameter)and mountingmedium(ProLongGold,Invitrogen,Carlsbad,USA),and sampleswereimagedbyfluorescence microscopyusinga Zeiss Axiovert200(Zeiss,Göttingen,Germany)witha20×objective.
2.8. Statistics
Culturesgrowingonpatternedislandswereassessedbyphase
Culturesgrowingonpatternedislandswereassessedbyphase