DPMwww.hhh258

AbstractTheFibra-Celpacked-bedbaskettechnologyhasbeenestablishedasanexcellentmethodforthegrowthofsuspensionandanchorage-dependentcelllines.
ThethreedimensionalstructureoftheFibra-Celdiskprovidesanexcellentsolid-supportmatrixfortheentrapmentorattachmentofanimalcells,allowingconstantperfusionofnutrientsinalow-shearenvironment.
Inthisapplicationnote,weshowthatHybridomacellscanbesuccessfullycultivatedinhighdensitiesinthe2.
5Lpacked-bedFibraCelbasketcontrolledbyaCelliGen310bioreactor.
HybridomacultureusingNewBrunswickCelliGen310withPacked-bedFibra-CelBasketImpellerRayRose,StaceyWillard,MaSha,EppendorfInc.
,Enfield,CT,U.
S.
A.
IntroductionPacked-bedbioreactorcellcultureisgenerallyacceptedasoneofthebestmethodstosimulatetheconditionsofanimalcellgrowthinvivosincecellsaremaintainedinalow-shearenvironmentwithconstantrefreshmentofnutrientsandremovalofwaste.
Thegrowthofattachment-dependentcellsonFibra-Celhasbeenshowntoincreasebothcellandproductyields.
Inparticular,Hybridomacellsareinherentlysensitivetowastebuildupandtheimplementationofpacked-bedFibra-Celgrowthconditionsinadditiontoperfusionproductionmethodshasgreatlyincreasedyields.
TodemonstratethattheCelliGen3102.
5Lbasketimpellerbioreactoriscapableofrobust,reproduciblehighdensityHybridomacultureunderperfusionconditions,twoindependenttrialswereconductedusingthesuspension-adaptedDA4.
4hybridomacellline.
MaterialsandMethodsInoculumpreparationDA4.
4Hybridomacells(ATCC#HB-57;Manassas,VA)weregrownin1Lshakeflasksat37°Cwith5%CO2andagitationsetat95rpm.
CulturemediumwaspreparedusingGibcoHybridoma-SFMcompleteDPMpowdersupplementedwith5%HycloneFetalBovineSerumand1%GibcoliquidPen/Strepbeforesterilefiltrationusinga0.
2mMilliporeMillipakgammagoldfilterintosterileHyclonebags(5Land10L,asnecessary).
Mediumwasstoredat2–8°Cuntiluse.
The1.
75Lvesselworkingvolumewasinoculatedwithatargettotalof4.
1x108cells.
Actualviablecellnumberswere3.
5x108cells(2.
2x105cells/mL)forthefirstrunand4.
8x108cells(3x105cells/mL)forthesecondrun.
Thetablebelowshowstheoriginofthematerialsusedinthisstudy.
MaterialSupplierCatalog#Lot#Hybridoma-SFMcompleteDPMpowderGibco12300-067949234Pen/Strep100XliquidGibco151401092590HycloneFetalbovineserumHycloneSH30070.
03AWC99936D(+)-GlucoseHybri-MaxpowderSigmaG5146-10k071M01453V45%GlucosesolutionSigmaG876954K2371Fibra-CelEppendorfM1292-9988Trial1:78690Trial2:1100081APPLICATIONNOTENo.
AA258IJuly2013Figure1.
Left:Thepacked-bedbasketimpellerincludingFibra-Celdisks.
Right:TheCelliGen310bioreactorwith2.
5Lvessel.
APPLICATIONNOTEINo.
AA258IPage2BioreactorconditionsForbothruns,hybridomacellswereculturedinthesamevessel,usingthesameCelliGen310cabinetfor9consecutivedays,usingthebasketimpellersystempackedwith75gofFibra-Celdisks.
CelliGen310SetpointsAgitation80rpmTemperature37°CpH7.
15Deadband0.
04DO50%Gassupplied4-gasmixcontrol(N2off;CO2fopHcontrol)Gasflowconditions0.
4SLPMVessel2.
5LglasswaterjacketedFibra-Cel75gPerfusionwasinitiatedforeachbioreactoronday3andcontinuedthroughday9.
Initially,themainobjectivewastoincreasetheperfusionratetomaintainaglucoseconcentrationaboveandnear1g/L.
Forthesecondbioreactorexperiment,theperfusionratewasadjustedtomatchthefirstbioreactorrateinordertomakethetworunsasidenticalaspossible.
Thetablesillustratetheexperimentalparametersandperfusionvolumesforbothtrials.
DayPerfusionvolume(L)30.
7341.
8154.
2565.
574.
2584.
7595BiochemistryanalysisDailyoff-linemeasurementsofglucoseconcentrationwereperformedusingaYSI2700analyzer(YSI,Inc.
,YellowSprings,OH).
Theglucoseconsumptionwascalculatedforeachtimepointandplottedasanaverageofthetwoindependenttrials.
Errorbarsindicatestandarderrorofthemean.
ResultsandDiscussionAspresentedinthegraphbelow(Figure2),therateofglucoseconsumptionacrossbothtrialsisindicativeofreproduciblegrowthofhybridomacellsinthisenvironment.
WeconcludethattheuseofFibra-CelinthebasketimpellersystemontheCelliGen310isanexcellentmethodforhighdensityhybridomaculture.
InabatchrunwiththeCelliGenpitchbladebioreactor,hybridomacellsusuallypeakatapproximately5g/dayofglucoseconsumption.
Thepacked-bedbasketimpellersystempresentssignificantlyhigherproductivitywithglucoseconsumptionpeakingat,onaverage,25g/day.
Inaddition,ifgrowthconditionsaremaintainedbycontinuedfreshmediaperfusionandglucoseconcentrationisneverallowedtofallbelow1g/L,hybridomacanbecontinuouslyculturedinthebasketmanydaysafterthe9daywindowprovidedinthisstudy;thisfurtherincreasesproductivityanddecreasesoverallantibodyproductioncosts.
Nooptimizationofgrowthconditionswereattemptedforeitherbioreactorrun.
302520151050Glucoseconsumption(g)Averageglucoseconsumption0246810DayFigure2.
Theglucoseconsumptionwascalculateddailyforeachbioreactorandthemeanispresented.
Errorbarsindicatestandarderrorofthemean.
Comparableconsumptionwasobservedacrossthetwobioreactors.
www.
eppendorf.
comATCCisaregisteredtrademarkofAmericanTypeCultureCollectionCorp.
,Virginia,USA.
HycloneisaregisteredtrademarkofHyCloneLaboratories,Inc.
,Utah,USA.
Fibra-CelisaregisteredtrademarksofImerysMineralsCalifornia,Inc.
,USAandlicensedtoNewBrunswickScientificCo.
,Inc,USA.
GibcoisaregisteredtrademarkofLifeTechnologiesCorporation,Delaware,USA.
MillipakandMilliporeareregisteredtrademarksofMerckKGaA,Darmstadt,Germany.
CelliGenisaregisteredtrademarkofNewBrunswickScientific,USA.
SigmaisaregisteredtrademarkofSigma-Aldrich,Co.
,LLC,USA.
YSIisaregisteredtrademarkofYSI,Inc.
,YellowSprings,USA.
EppendorfandtheEppendorflogoareregisteredtrademarksofEppendorfAG,Germany.
NewBrunswickisatrademarkofEppendorfAG,Germany.
Allrightsreserved,includinggraphicsandimages.
Copyright2013byEppendorfAG,Germany.
Yourlocaldistributor:www.
eppendorf.
com/contactEppendorfAG22331HamburgGermanyE-mail:eppendorf@eppendorf.
comwww.
eppendorf.
comAPPLICATIONNOTEINo.
AA258IPage3OrderinginformationDescriptionNorthAmericaOrderno.
InternationalOrderno,NewBrunswickCelliGen310Bioreactor,2.
5LSystemM1287-1260(100-120V)M1287-1264(200-240V)Fibra-CelDisks,250gramsM1292-9988M1292-99882.
5LBasketImpellerKitM1287-1140M1287-1140