三维细胞培养和二维培养的优点.pdf

三维细胞培养和二维培养的优点.pdf

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Reportswww.BioT325Vol.52|No.5|2012Thereisampleevidencethat3-Dcellculturesbettermimictheinvivoconditionsofamulticellularorganismthando2-Dcultures,inwhichcellsadheretoglassorplasticsurfaces（14）.Often,theresponsesofcellsina2-Dculturetovariouscuesarequitedissimilarfromthoseofthecellsinvivoorina3-Dculture（58）.Thisispartlybecausethephysiologyofacellisdeter-mined,inadditiontothegenome,bythemicroenvironment,includingmechanicalpropertiesoftheextracellularmatrix（ECM）andphysicalandchemicalanisot-ropies（2,911）,whicharequitedifferentin3-Dand2-Dcultures（7,12,13）.Therefore,3-Dculturesarepreferableover2-Dfortheuseinanumberoffields,suchasstudiesonstemcelldifferentiation,tissuemorpho-genesis,cancerbiology,cell-virusinterac-tions,andcell-baseddrugscreeningandtoxicologyassays（1,14,15）.Still,2-Dculturescultureshavetheadvantageofpresentingeverycellinthesameplane.Thisensuresthatallthecellsareculturedunderidenticalconditions,includinggasexchange,nutritionsupplyandwasteremoval,andpermitsthecellstobeeasilymonitored,screened,andcollectedforfurtheruse.Onthecontrary,ina3-Dculture,cellslocatedatdifferentdistancesfromthesurfaceencounterdifferentphysi-ologicalconditions（3,8）.Avarietyofsophisticatedandexpensivesystemshavebeendevelopedtominimizesuchchemicalgradients（3）.Anotherproblemisthediffi-cultyofusingconventionalmicroscopesformonitoringcellsimmersedatdifferentdepthsinahighlyscatteringmedium（7）.Boththeproblemswerepartiallyovercomeby“on-top”cultures,inwhichcellsareculturedinaliquidmediumwhilebeingattachedtoasurfaceofagel,whilemimickingtosomeextenttheconditionsofa3-Dculture（16,17）.However,insuchaformat,cellsandtheirprogenyarenotsecurelyimmobilizedandmayoccasionallymigrateintothesurroundingsolution.Also,beforetheybecomeattachedtothegelsurface,cellstendtoaggregatewithoneanotherandmayunevenlyspreadoverthegelsurface（17）.Herewereportunordered2-Darraysofeukaryoticcellsthatcombinetheadvan-tagesofthemicroenvironmentofa3-Dculturewiththeuniformityofconditionsandeaseofobservationcharacteristicofa2-Dculture.Thisplanar3-Dculturemaybeofuseinanumberofresearchandappliedfieldsrequiringobservation,manip-ulation,andproliferationofalargenumberofindividualeukaryoticcellsunderstrictlycontrolledconditions.MaterialsandmethodsAdherentcellsHeLa（CCL-2;ATCC,Manassas,VA,USA）,HEK-293（CRL-1573）,H1299（CRL-5803）,andSC-1（CRL-1404）weregrownto90%100%confluencein25cm2flasksanddispersedaccordingtotheATCCprotocol（www.lgcstandards-atcc.org）followedbytheadditionof10mLDulbeccosmodifiedEaglesmedium（DMEM）supplementedwith10%FBS（PAALaboratories,Pasching,Austria）,4mMglutamine,50U/mLpenicillin,and50g/mLstreptomycin.SuspensioncellsDT40（CRL-2111）weregrowninDMEMsupplementedwith8%FBS,2%chickenserum（cat.no.C5405;Sigma-Aldrich,St.Louis,MO,USA）,2mMglutamine,50M2-mercaptoethanol,50U/mLpenicillin,and50g/mLstreptomycinuntilthedensityof106cell/mL,whereascellsK-562（CCL-243）weregrowninRPMI-1640mediumsupplementedwith10%FBS,4mMglutamine,50U/mLpenicillin,and50g/mLstreptomycinuntilthedensityof0.5106cells/mL.FortransfectingHEK-293cells,0.3gGFP-encodingplasmidpEGFP-C3（ClontechLaboratories,MountainView,CA,USA）and1LUnifectin-56（UnifectGroup,Moscow,Russia）weremixedwith225Lserum-freeDMEM,incubatedfor20minat22C,andaddedtoawellofa12-wellplatecontaining1-day-old50%70%confluentcelllayerunder0.9mLofthecompletegrowthmedium,andthecellsweregrownforonemoredaybeforeuse.Planararrangementofeukaryoticcellsinmergedhydrogelscombinestheadvantagesof3-Dand2-DculturesAlexanderA.Gordeev,HelenaV.Chetverina,andAlexanderB.ChetverinInstituteofProteinResearchoftheRussianAcademyofSciences,Pushchino,MoscowRegion,RussiaBioTechniques52:

325-331（May2012）doi10.2144/000113861Keywords:

suspensioncells;adherentcells;populationstudies;cellimmobilization;high-throughputscreening;celllines;cellcloningSupplementarymaterialforthisarticleisavailableatwww.BioTreportanunordered2-Darrayofeukaryoticcellscompletelyembeddedina3-Dmatrix.Everycellislocatedatthesamedistancefromthegelsurface,whichensuresuniformityofgrowthconditionsandeaseofobservationcharacteristicofa2-Dculture.Eachcellisfirmlyimmobilized,andeachhasauniqueaddressinthearray.Thecellscanberapidlyscreened,individuallymonitoredduringextendedtimeperiods,andculturedwiththeformationofspheroidmicrocoloniescharacteristicofa3-Dculture.Individualmicrocoloniescanbeextractedfromthegelandfurtherpropagated,thusenablingisolationofpurecellclonesfromratherdensecellpopulationsandrapiddrug-freegenerationofstablecelllines.ReportsReportswww.BioT326Vol.52|No.5|2012Polyacrylamide（PAA）gelswerecastin14-mm-diameter,0.4-mm-deepwells,thenwashedanddriedasreported（18）.Mergedgelswerepreparedasfollows.Cellswerepelletedinacentrifugefor5minat200g（or300gforDT40）,resuspendedinDulbeccosphosphate-bufferedsolution（DPBS;8mMNa2HPO4/1.5mMKH2PO4,pH7.5,138mMNaCl,2.7mMKCl,0.9mMCaCl2,0.5mMMgCl2）tothedesiredconcentration,andincubatedfor2minat30C.Thecellsuspensionwasmixed（1:

1,v/v）withacooledto30Cmolten1%agarose（TypeIX,Ultra-lowGellingTemperature,cat.no.A5030;Sigma-Adrich）inDPBS,and70LmixturewerepouredintoawellcontainingdryPAAgelfilmattachedtoitsbottom,withsimultaneousslidingacoverslipoverthewell.Incaseofinvertedmergedgels,thePAAgelwasattachedtothecoverslip.Whereindicated,theslidewasspunfor1minat100g,25Cincentrifuge5804R（Eppendorf,Hamburg,Germany）,byplacingtheslidewiththeagaroselayerfacinguponthetubeadapterofabucketrotor（cat.no.A-444;Eppendorf）.Theagarosewasthensolidifiedat4Cfor20min.Cellswereinspectedusingwide-fieldinvertedmicroscopeDMIRE2withmechanicalstage,integratedintotheASTPsystem（LeicaMicrosystemsGmbH,Wetzlar,Germany）,ineitherabrightfieldmodeusingtheLeicamodulationcontrastorinaGFPfluorescentmodeusingtheI3filtercube（excitationfilterBP450/490,dichromaticmirror510,suppressionfilterLP515）.ImageswereobtainedusingeitherCoolpi4500photocamera（NikonRussia,Moscow,Russia）orCascadeII512videocamera（Photometrics,Tucson,AZ,USA）.OpticalsectionsandsideviewsoffluorescingcellsandmicrocoloniesintheGFPfluorescentmodewereobtainedusingconfocalmicroscopeTCSSPEDM2500andLASAFVersion2.1.0build4316software（Leica）.Whereindicated,non-GFPproducingcellswerestainedwithabroadrangefluorescentinkbyspottingthesideofthecoverslipfacingthegelwithaCD/DVD/BDmarker（LINER2616;Centropen,Prague,CzechRepublic;www.centropen.cz/product-catalogue/for-the-office/12-3-cddvdbd-markers/38-cddvdbd-liner-2616）.Microcoloniesweregrownininvertedmergedgelswhoseagaroselayercontainedanappropriatecompletegrowthmediumwithorwithout0.5mg/mLbovineskincollagen（cat.no.C4243;Sigma-Aldrich）.Thegelswereincubatedupsidedownina40-mmPetridishat37Cand5%CO2under3mLgrowthmedium.Microcol-onieswereextractedbysuckingwithadisposable70-minnerdiameterglassmicropipetmadefromaborosilicateglasscapillarytubing（cat.no.BF100-50-10;SutterInstrumentCompany,Novato,CA,USA）usingP-97MicropipetPuller（SutterInstrument）.ThemicropipetwasoperatedwiththeCellTramAirmicro-injector（EppendorfAustriaGmbH,Vienna,Austria）mountedontheTransferManNK2micromanipulator（Eppendorf）integratedintotheLeicaASTPsystem.ResultsanddiscussionWeprepared2-DcellarraysusingmergedPAA/agarosegels（1819）.Tothisend,asuspensionofcellsinmoltenlow-gellingtemperatureagarosewaspouredinashallowwellmadeinamicroscopeslidehavingapreviouslycastanddriedpolyacrylamidegelcovalentlyattached.WhenthePAAgelswellsbyabsorbingtheliquid,itdisplacescellstowardthecoverslip.Uponformation,theagarosegelbecomespartlymergedwiththePAAgelandimmobilizescellsconcentratedonthePAAgelsurface.Sometimes,especiallyfortheexperimentsoncellgrowthandcloning,weusedinvertedmergedgels.Inthiscase,thedryPAAmatrixwascovalentlyattachedtoaglassslipcoveringawellfilledwithmoltenagarosecontainingcells.Tobecomeimmobilized,cellsmustbesubmergedintheagarosegelatsomedepth;hence,theagarosemusthardenbeforeitiscompletelysuckedupbythePAAmatrix.However,inthiscase,cellsarenotlayeredintoamonolayer;rather,theypopulatetheentirespaceabovethePAAsurface（Figure1A）.Wefoundthatanearlyperfectmonolayercanbegeneratedbybrieflyspinningtheslideinacentrifugewhiletheagaroseisstillliquid;thisforcesallthecellstosedimentontheswellingPAAbed.WhenthePAAgelwasmadeof7%acrylamideand0.07%bisacryl-amide,thecellsweresubmergedatadepthof10m（Figure1B）.Decreasingtheacryl-amideconcentrationto4%andincreasingthebisacrylamide/acrylamideratioto1/20increasedthedepthto25and20m,respectively（SupplementaryFigureS1）.Apparently,thelowerpercentageofacryl-amidemakesthePAAgelmorecompressibleduringcentrifugation,whereasthehigherpercentageofthecross-linkerreducestherateofPAAswelling.Theresulting2-DarrangementallowsallthecellswithinaFigure1.HeLacellsinmergedgels.Cellssuspendedinmolten0.5%agarosewerepouredintoawellmadeinamicroscopicslide,thewellbottombeingcoveredwithadryPAAgel,followedbycoolingtheslideonice.Shownareconfocalmicroscopeimagesoffluorescentlystainedcells.（A）Sideviewofacelllayerformedwithoutcentrifugation.（B）S