3D细胞培养支架资料

3维细胞培养技术在干细胞和药物开发中的应用上海复蒙基因生物科技有限公司上海市邯郸路100号复旦大学创新创业基地3DBiotek,LLCNorthBrunswick,NJ08902HistoryofCellCulture1838Schleiden&Schwann“celltheory”1907HarrisonNerveoutgrowthInventoroftissueculture1955Eagledefinedmedium1981Martin&EvansMouseEScells3D1885WillhelmRouxcellscanliveoutsidethebody1952GayEstablishedHeLacells1965HamColonialgrowthofmammaliancells1998Thomson&GearheartHumanEScells1665Hookediscovered“cells”NewConferenceon3DCellCultureEngineeredInvitroTissueModelforDiseaseandDrugDiscoveryStudiesGeneralSession2010AnnualConferenceSocietyforBiomaterials(USA)April21-24,2010_______________________________________________________AdheringCellCultureLimitationofCurrent2DCellCultureTechniqueEmbryonicFibroblastsHeLaCellsHumanEmbryonicStemCellsLimitation:2Dcellcultureconditiondoesnotmimicrealliving3Dcellgrowthconditioninvivo.StemCell/TissueEngineeringApplications•3Dporousscaffoldsareneededforboneandcartilage…NeedFor3DScaffoldstissueengineeredbladder…Dr.AnthonyAtala,WakeForestUniversityinNorthCarolina=1800727&page=12006Needfor3DPorousScaffoldsforcreateanewheartintheLab…Current3DScaffoldsontheMarket_________________BD™3DCalciumPhosphateScaffoldBD3DCollagenScaffold__™________™_______BD3DOPLAScaffoldNotwidelyaccepted/usedduetolimitationsWellDefined3DScaffoldsFabricatedusingPrecision3DMicro-fabricationTechnology•100%openporosity•Well-definedporesizeandporousstructure•Organicsolventfree•CustomdesignandfabricationPolycaprolactonescaffoldsPolycaprolactone(PCL)isabiodegradablepolymerusedinFDAapprovedmedicaldevicesAC3DPCLScaffoldsControlledporesize:BPCLscaffold.(A-B)Topviewand(C)ScanningElectronMicroscopy(SEM)characterizationofPCLscaffolds.200~500µmControlledstrut:200~500µmStemCellCulture/differentiation3DPCLScaffoldsDay1afterhMSCdifferentiation,10xDay7afterhMSCdifferentiation,10x3DbiodegradablePCLscaffoldssupporthMSCgrowth.Aftertheonsetofobsteoblasticdifferentiation,hMSCsweremonitoredusingalightmicroscope.StemCellCulture/differentiation3DPCLC.Figure2.OsteoblasticdifferentiationofhMSCsculturedon3DbiodegradablePCLscaffolds.After3weeksofculturein10%growthmediawith(A-B)VitaminDorwiththe(C)osteogenicfactorsdexamethasone,ascorbicacid,and_-glycerolphosphate,hMSCswerestainedwithToluidineblueandembeddedinTechnovit.StemCellCulture/differentiationVonKossastaining3DPCL0.450.40.350.30.250.20.150.10.052D3DMTTassay0Day13123DPCLscaffoldsupportsstemcelldifferentiation.Humanmesenchymalstemcellswereculturedin2Dandon3DPCLscaffolds.VonKossastainingwasperformedin2D(toppanels)andon3DPCLscaffolds(bottompanels)ondays7and28ofstemcellculture.Redarrowsindicatepositivestainingformineralizednoduleformation.ViabilitywasmeasuredwithMTTassay.Absorbance(570nm)___________________________________________________________!#_$_%Enhancementofcellularresponsesand5integrindependenceincell-derived3Dmatrices(A)Fibroblastattachmenttotheindicatedsubstrates.(B)Typicalmorphologiesofcellsondifferentsubstrates.Notethatfibroblastsin3Dmatrixachievedtheirfinal,elongatedmorphologyby5hours(3Dmat,control)(C)Humanfibroblastmigrationtracedbytime-lapsevideomicroscopy(26).Scalebars,100µmNATURE,VOL424,21AUGUST2003Rolereverse:unlikein2-Dculture,breasttumourcellsin3-Dculture(left)thatbecomemalignant(center)canbemadetoreverttotheiroriginalstate(right)whenanantibodyagainstB-integrinisaddedtothesystemDr.MinaBissell,LawrenceBerkeleyNationalLaboratory,California,USATheNeedforConvenientandEasytoUse3DScaffoldsExpandtheuseof3DscaffoldstoDrugdiscoveryandTumorModelsCellBasedScreeningInDrugDiscovery•TargetValidation•Primaryscreening•Secondaryscreening(hittolead)•ADMET(adsorption,distribution,metabolism,excretionandtoxicity)DevelopmentofANovel3DPSScaffoldIdeal3DScaffolds1.Non-degradable2.Compatiblewithmost2Dassays3.Easymonitoringcellgrowthwithinthescaffolds4.Acceptablenon-cytotoxicmaterialUniquenessofPolystyrene(PS)ScaffoldsSameTissueCulturePlastic,Nowin3D!!!ABCFour-layerstructuraldesignofPSscaffold.Fourdistinctlayersarevisiblefrom(A)side-angle,(B)side,and(C)top.SameTissueCulturePlastic,Nowin3D!!!TissueCulturePlastics:2Dvs3DAtday4,F-actinisformedinsheetson3DPSscaffolds.NIH-3T3cellswereplatedon(A-B)a96welltissuecultureplateatadensityof9.6x10^2cells/cm2oron(C-D)a1520wellPSscaffoldatadensityof3.2x10^3/cm2.Onday4afterseeding,cellnucleiandF-actinmicrofilamentswerestainedwith(A,C)DAPI(blue)and(B,D)AlexaFluor488phalloidin(green),respectively(50X).Day4PolarizedCellsin3DCultureAtday7,F-actinisorganizedalongthedirectionofthefiber.NIH-3T3cellswereplatedon(A-B)a96welltissuecultureplateatadensityof9.6x10^2cells/cm2oron(C-D)a1520wellPSscaffoldatadensityof3.2x10^3/cm2.Onday7afterseeding,cellnucleiandF-actinmicrofilamentswerestainedwith(A,C)DAPI(blue)and(B,D)AlexaFluor488phalloidin(green),respectively.RedarrowsindicatedirectionofF-actinfilamentsalongasinglePSfiber(40X).Polarized3DStructureFibroblastsform3Dtissuelikecell/ECMstructurewithinthescaffold.F-actinfilamentsformattachments(A,B)betweenindividualfibersand(C,D)atthefiberjoint.CellnucleiandF-actinmicrofilamentswerestainedwith(A,