BD-3D细胞培养手册

BDBiosciencesFebruary16,20113DCellCultureSystemsMarshallJ.Kosovsky,Ph.D.2TopicsforDiscussion•OverviewofCellCultureSystems:2Dvs.3D•ExtracellularMatrix(ECM)Proteins–Epithelialcelldifferentiation–Endothelialcelltubeformation•Animal-FreeMatrix:BD™PuraMatrix™PeptideHydrogel–Primaryhepatocytesdifferentiation–Neuronalcelldifferentiation–Endothelialcelltubeformation•CellCultureInsertPlatform–Tumorcellmigration/invasion–Endothelialcellmigration/invasion–Generalcelldifferentiation(3Dco-culture)3Cellculturesystems:2DÆ3D2D2D¾Non-treated(hydrophobic)¾BDFalcon™TissueCulture(TC)-treated[net(-)charge]¾BDPrimaria™[mixtureof(-)and(+)charge]¾BDBioCoat™(largevarietyofECMs,poly-lysine)¾BDPureCoat™[carboxyl(-),amine(+);animalfree]3D3D¾ECMcoatings(BDMatrigel™matrix,BD™Laminin/Entactin,Collagens)¾Rigid3DScaffolds(e.g.,PLA,PGA)¾Animal-freehydrogels(BD™PuraMatrix™PeptideHydrogel)¾Cellcultureinserts(e.g.,co-culturemodelssuchasBBB)¾Animalmodels42Dvs.3DCellCultureYesNoCellEncapsulationSlow;biochemicalgradientsregulatecell-cellcommunicationandsignalingRapidGrowthFactorDiffusion‘Physiological’;promotescloseinteractionsbetweencells,ECMs,growthfactorsNotphysiological;cellspartiallyinteractArchitectureMimicsnaturaltissueenvironmentRigid;inertGrowthSubstrate3D2D5WhyCultureCellsin3D?•Tissueandorgansexistin3D•Studyingcellsin3Denablesresearchersto‘mimic’orapproximatephysiologicalconditionsthatexistinvivo•Stronghistoricalevidencethat3Dcellcultureworksforestablishingdifferentiatedcellmorphologyandfunction__________________________________________________________________FeatureArticlesinNature,August2003Goodbye,FlatBiology?....biologistsstartingtoexplorethemeritsofculturingcellsin3Dhavebeenstunnedbythedifferenceitmakestothewaycellsbehave,whichismuchclosertotheirbehaviorinvivo.Biology’sNewDimension(highlightsBDMatrigel™matrixandBD™PuraMatrix™)“In10years,anyonetryingtouse2Danalysestogetrelevantandnovelbiologicalinformationwillfinditdifficulttogetfunded.”–MihaelPolymeropoulos,CSO,VandaPharmaceuticals(formerHeadofPharmacogenetics,Novartis)6CellCultureModelsSupportorPromoteCellBehaviorCellbehaviors(differentiation,functionality)influencedbycuesinthemicroenvironment:•Cellmorphology(structure,phenotype)•Polarity(functionaldirectionality)•Growth(proliferation)•Cellmotility(migration,invasion)•Neuriteoutgrowth•Signaltransduction(surfacereceptorfunction)•Geneandproteinexpression(differentcelltypescanexpressdifferentgenes/proteins;livervs.heartvs.brain)•Biochemicalactivities(proteins,enzymes)7TheExtracellularMatrix•Complexmixturecontainingglycoproteins,collagens,andproteoglycans•Formsstructuralframeworkthatstabilizestissuesandprovidesmechanicalsupportforcellattachment•Playsimportantroleincellfunctionalityanddifferentiation–Receptor-mediatedsignaling–Regulationofgeneexpression8basallamina=basementmembraneBDMatrigel™matrix=reconstitutedbasementmembraneFigure:MolecularBiologyoftheCell(3rdEdition)TheBasalLamina:AThin‘Mat’thatUnderliesEpithelialCellSheetsandTubes9epithelialcellsbasallaminacollagenfibrilsFigure:MolecularBiologyoftheCell(3rdEdition)BasalLaminainChickEmbryoCornea10ECMContributestoIntracellularSignalingPathways•ECM-basedgrowthsubstratesprovideaphysiologicalenvironmentthatsupportsandpromoteskeycellfunctions•ECMmoleculesinteractwithcellsurfacereceptors(e.g.,regulationofintegrinsignalingbyfibronectin:integrininteractions)•ECMappearstofunctioninthestorageandpresentationofgrowthfactors11CompositionLaminin~60%CollagenIV~30%Entactin~8%Heparansulfateproteoglycan(perlecan)Growthfactors(e.g.,PDGF,EGF,TGF-β)MatrixmetalloproteinasesBDMatrigel™matrix:ReconstitutedBasementMembrane12BDMatrigelMatrixHighConcentration•Proteinconcentration:~18-22mg/ml•Applications¾Humantumorxenograftinimmunodeficientnudemice−SubcutaneousinjectionofMatrigelmixturecontaininghumantumorcells(e.g.,lung,prostate,breast,colon)¾‘BDMatrigel™matrixPlugAssay’forinvivoangiogenesis−SubcutaneousinjectionofBDMatrigelmatrixmixturecontainingtestsubstances(e.g.,antibodies,growthfactors,syntheticpeptides)and/ortumorcells−Removalofplugandanalysisofnewvesselformation13HighConcentrationECMProteinsfor3DCellCultureHighConcentrationLaminin/EntactinComplex•MajorcomponentofbasementmembraneinEngelbreth-Holm-Swarm(EHS)mousetumors•Proteinconcentration:≥10mg/ml•Purity90%bySDS-PAGE•Forms3Dgelthatmodelstissuemicroenvironmentinvivo•Supportscelldifferentiation(e.g.,mousesubmandibularcells,endothelialcelltubeformation)HighConcentrationCollagenI•Source:rattailtendon•Proteinconcentration:8-11mg/ml•Full-lengthprotein(nottreatedwithpepsin)•Purity90%bySDS-PAGE•Formssturdygelthatprovidesmaximal3Dsupportmatrix14FactorsthatInfluenceCellDifferentiationandFunctionality•Biologicalcompositionofthecultureenvironment(e.g.,celltypes,ECMs,growthfactors)•Molecularinteractionsandcelladhesion(cell:cell,cell:ECM,ECM:ECM,cell:growthfactor,andECM:growthfactor)•Mechanicalstrengthandstructuralproperties(degreeofrigidity,3Darchitecture)15CollagenI(2Dthincoat)CollagenI(3Dgel)BDMatrigel™matrix(3D)PrimaryHepatocytesExhibitDifferentiatedMorpholog