生物反应器教学课件DifferentcellpropertiesSelectcellculturemethod

ContentIntroductionCellculturespecificationSelectionofbioreactorMicroorganismsBacteria(0.5–2µm)Escherichiacoli(lowerintestine)Yeast(2–5µm)Saccharomycescerevisiae(baker’syeast)Fungi(2–15µm)Penicilliumspec.(Cheeseandantibiotic)Mammalian(7–20µm)ChineseHamsterOvaryCells(Insuline)Animalcellsvs.microbialcellsPROTEINS-PosttranslationalmodificationsGlycosylation,phosphorylationetc.ProperfoldingenvironmentEfficientsecretionExtra-cellularmatrix–mediumforcellstointeractandmigrateAnchoragedependantorindependantAnimalcellcultureortheabilitytocontinuouslyculturecellsPlatformtoinvestigatenormalphysiologyandbiochemistryTesttheeffectsofdrugsandcompoundsinvitroTissueengineeringSynthesizevaluableproductsModelsforstudyingdiseasesCellStructureWidevarietyofcelllinescultured(epithelial,muscleetc.)Largerthanmicrobialcells(10-30m)ManyinternalorganellesNocellwall–susceptibletoshearforcesCytoskeleton–divisionand migrationAnchoragedependantCommoncelllinesandtheirapplicationsCellGrowthconditionsGrowthtemperatureMammalian37CInsect27CDissolvedoxygenMediarequirements(insect,mammalian,serumetc.)BasicMediaDiffer

ForMammalianandInsectCultureComponentMammalianInsectGlucose4g/l2.5g/lAminoacids0.01-0.05g/l0.1-0.5g/lGlutamine1g/l1g/lHCO33.5g/l0.35g/lH2PO40.1g/l1g/lSalts4.5g/lNaCl1g/lMg2SO4,KClVitaminsMoreLesspH7.26.4Osmolarity300mOsm350mOsmSerumAdditionUsedat0-20%SerumItcontains:GrowthfactorsTransferrin(Fe)LipidsInsulinAlsoimportantfor:ShearprotectionDetoxificationProblems:Infectiousagents(viruses,mycoplasm,prions)Serumcompositionispoorlydefinedandthebatchesvary.ExpensiveGrowthcurvesinyeastsandmammaliancellsaredifferent0.000010.00010.0010.010.11.010.0050100

YeastMammalianMinimumdensityLagphaseExponentialStationaryDeclineCellMetabolismToxicmetabolitesLactatefromglucosemetabolism(lowerspH)AmmoniafromcatabolismofglutamateandaminoacidsTypeofcultivationBATCHFERMENTATION.Duration:0.5-1weeksCelldensity4*106c/mlFinish:nomorenutrientsTypeofcultivationFED-BATCHAdditionofconcentratednutrients=>higherproductconcentrationDuration:1-1.5weeksCelldensity5*106c/mlFinish:Viability<50%TypeofcultivationFED-BATCHContinuousadditionoffeed andcollectingofharvestDuration:1-3monthsCelldensity3*106c/mlTypeofcultivationPerfusionContinuousadditionoffeedandcollectingofharvestwithcellretentiondevice =>highercelldensityDuration:1-6monthsCelldensity20*106c/mlPerfusionUsedtoachievehighcelldensitiesFreshmediumaddedasspentmediumisremovedCellseparationrequired(spinfilters,hollow-fiberfilters,centrifugalseparation,BioSep)RemovetoxicsAdditionalconsiderationswithabioreactor?Noanchorage–CarriersadapttosuspensionShear–Reactordesign,PluorinicF68Wasteaccumulation– PerfusioncultureMicrocarrierculturesMicrocarriersprovideagoodsurfaceareforattachmentpervolume.FrequentlyusedwithsuspensionculturesofanchoragedependantcellsSolidmicrocarriers–sufferfromfluidmechanicaldamageMacroporousmicrocarriers–haveincreasedsurfacearea(gelatin,collagen,cellulose,polystyrene,polyethyleneetc.)MicrocarrierculturesPackedbed HollowfiberSuspensioncellsEasierscaleupAerationandAgitationStirringprovides:SuspensionofcellsandsolidnutrientsDispersionofimmiscibleliquidsEqualizationoftemp.andnutrientconc.DispersionofairAerationandAgitationOTR=KLa(C*-CL)CL=concentrationofdissolvedoxygeninthefermentationbrothC*=isthesaturateddissolvedoxygenconcentrationa=isthegas/liquidinterfaceareaperliquidvolumeKL=isthemasstransfercoefficientBioreactordesignAgitationSealsMechanicalFaceSealsLipSealsMagneticDrivesImpellersTurbineMarinePitchedBladeOthersAgitationAgitation-turbineimpellerForunaeratedwater20Cinafullybaffledtankthefollowingformulaapplies:P=4.5x10-13*Di5*N3*F Di=ImpellerDiameterinInch N=RPMofShaft F=One(1)foroneimpeller (1.8)fortwoimpellers(2.4)forthreeimpellersAgitation-turbineimpellerTipSpeedshouldalsobeconsideredbecauseoftendencytoshearcells.Donotexceed1m/sforcellcultureAnd5-6m/sformicrobialcultures.BioreactordesignconsiderationsShapeASMEBottomsHemisphericalBottomsJacketDesignConventionalDimpledHalfPipePortsTri-Clamp,DN,NA-connect,IngoldType,FlangetypeBioreactordesignconsiderationsASMEHead(s)HemisphericalHead(s)TemperaturecontrolTemperaturecontrolCoolingSystem:remove50to100Watts/LiterofVolume.HeatingSystems:One(1)degreeCperminutebetween25and45CSpargersRingTypeSingleOrificeSinteredSterilityConsiderationsKillingRatesDeadLegsFittingTypesPipingConsiderationsSterilityConsiderationsCriticalElementsofSteamSterilizationTime TemperatureMoisture AirRemovalSterilityConsiderationsSterilityConsiderations–KillingrateF0=∫ti*10(Ti-121)/ZF0=Integratedamountoflethalitydeliveredduringacycle i.e.AcyclewithF0=17minuteshasalethalityequivalentto17minutesat121C.CIPconsiderationsSterile