Introduction Qualityisimportantinallaspectsoftissueculturesincethequalityofmaterialsusedi.e.mediaandotherreagents)willaffectthequalityoftheculturesandproductsderivedfromthem.Themainareasofqualitycontrolthatareofconcernfortissuecultureare: ReagentsandMaterials Apotentialsourceofcontaminationisreagentsandmaterials,inparticularbovineserumwhichhasbeenidentifiedasasourceofbovineviraldiarrhoeavirus(BVDV).PorcinetrypsinisalsoapotentialsourceofMycoplasmahyorhinis.Goodqualityreagentsandmaterialsareavailablefromnumerousmanufacturersoftissueculturemediaandsupplements.InadditionmanufacturersincludingSigmawillcarryoutarangeofqualitycontroltestsincludingscreeningformycoplasmaandBVDVandsupplyaCertificateofAnalysiswiththeirproducts.Thesestatetheproductandlotnumbersandformsavitalpartofrecordkeepingandtrackingofreagentsusedintheproductionofcellstocks.ItisadvisabletofurthertestkeyreagentssuchasFBStoensurethattheyare‘fitforpurpose’duetobatch-to-batchvariation. Manufacturersofsterileplasticware(flasks,centrifugetubes,Pipettes)designedfortissuecultureusearealsosuppliedwithCertificatesofAnalysisforeachbatchproduced,whichshouldbekeptforfuturereference. ProvenanceandIntegrityofCellLines Thesourcingofcelllinescanhaveanimportanteffectonqualitysincefreshlyimportedcelllinesareamajorsourceofcontamination.Theadvantagesofobtainingcelllinesfromarecognizedsourcesuchasaculturecollectionare: Oncecelllineshavebeenobtainedfromareputablesourceitisimportanttoimplementmasterandworkingcellbankingproceduresandtheassociatedqualitycontrolstepssuchasroutinetestingformicrobialcontaminantsandconfirmingtheidentityofcultures. AvoidanceofMicrobialContamination Potentialsourcesofcontaminationincludeothercelllines,laboratoryconditionsandstaffpoorlytrainedincoreareassuchasaseptictechniquesandgoodlaboratorypractice.Thustheuseofcellsandreagentsofknownoriginandqualityaloneisnotsufficienttoguaranteequalityofproduct(cellstockorcultureproducts);itisnecessarytodemonstratequalitythroughouttheproductionprocessandalsointhefinalproduct.Routinescreeningaidstheearlydetectionofcontaminationsinceallmanipulationsareapotentialsourceofcontamination. The3maintypesofmicrobialcontaminantsintissuecultureare: BacterialandFungalContaminationBacterialcontaminationisgenerallyvisIBLetothenakedeyeanddetectedbyasuddenincreaseinturbidityandcolorchangeoftheculturemediumastheresultofachangeinpH.Thecellculturemaysurviveforashorttimebutthecellswilleventuallydie.Dailymicroscopicobservationofcultureswillensureearlydetectionofcontaminationandenableappropriateactiontobetakenassoonasthefirstsignsofcontaminationbecomeapparent(seebelow).Inadditionspecifictestsforthedetectionofbacteriaandfungishouldbeusedaspartofaroutineandregularqualitycontrolscreeningprocedure(seeProtocol8). MycoplasmaContaminationMycoplasmasarethesmallestfree-livingself-replicatingprokaryotes.TheylackacellwallandlacktheABIlitytosynthesizeone.Theyare0.35mindiameterandcanbeobservedasfilamentousorcoccalforms.Thereare5majorspeciesthataretissueculturecontaminants,namelyM.hyorhinis,M.arginini,M.orale,M.fermentansandAcholeplasmalaidlawii. Theeffectsofmycoplasmainfectionaremoreinsidiousthanthoseofbacteriaandfungiinducingseverallongtermeffects.Theseinclude: However,despitethesewell-documentedeffectsthepresenceofmycoplasmaisoftennottestedforwiththeconsequencethatinsuchlaboratoriesthemajorityofcelllinesarepositiveformycoplasma.Mycoplasmacontaminationisdifficulttodetectrequiringtheuseofspecialisttechniques(seeProtocol9-IsolationbycultureandProtocol10–DetectionbyDNAstaining).Inthepastonlyspecialistlaboratories,suchasculturecollections,haveperformedthesetests.Howeveravarietyofcommercialkitsarenowavailablealthoughtheperformancecharacteristicsofthesekitscanbeextremelyvariable.Acombinationoftheseshouldbeusedaspartofaroutineandregularqualitycontrolscreeningprocedure.CulturecollectionssuchasECACCareabletotestculturesifrequired.MycoplasmatestingproductsareavailablefromSigma,refertopages489-490ofLifeSciencesCatalogue. ViralContaminationSomecelllinescontainendogenousvirusesandsecretevirusparticlesorexpressviralantigensontheirsurface(e.g.EBVtransformedlines).Thesecelllinesarenotconsideredcontaminated.However,bovineserumisapotentialsourceofbovineviraldiarrhoeavirus(BVDV)contamination.Useofinfectedserumwillleadtocontaminationofcelllineswiththevirus.ContaminationofcelllineswithBVDVmaycauseslightchangesingrowthratebutsincethisvirusisnon-cytopathicmacroscopicandmicroscopicchangesintheculturewillnotbedetected.SuppliersofbovineserumareawareofthisandscreenseraaccordinglyandgenerallyserumissoldasBVDVtested. EnvironmentalMonitoring Itisgoodpracticetomonitorthelaboratoryenvironmentwherecellculturesandtheirproductsareprepared.ClassIImicroBIOLOGysafetycabinetsshouldbecheckedevery6monthstoensurethattheyareworkingefficiently.Howeveritisalsoadvisabletomonitorthenumberofcontaminantswithinthecabinetbyperiodicallyplacingopensettleplates(bloodagarbacteriologicalcultureplates)onthecabinetworksurfaces.Inadditionsettleplatesshouldbeusedtoassessairbornemicrobialburdenatselectedpointsaroundthelaboratory.Platesshouldbeleftopenforaperiodof4hours.Afterthistimetheyshouldbecovered,placedinsealedboxesandincubatedat32ºCand22ºCforupto7days.Attheendofthisperiodtheplatesshouldbeexaminedforthepresenceofmicrobialgrowth.Thepositionofeachplateinthecabinetshouldberecordedandresultsstoredfortrendanalysis. Acceptablelimitsshouldbedefinedintermsof“alert”levelsand“action”levels,theactualvaluesbeingdependentonthecriticalityoftheworkandthelevelsofcleannessthatcanbeachievedundernormaloperatingconditions. Whattodointheeventofcontamination Onehugelyunder-estimatedproblemintissuecultureistheroutineuseofantibiotics.ContinuoususeofantibioticsisunnecessaryandcanleadtothedevelopmentofresistantstrainsthataredifficulttoerADIcateandmayrequiretheuseofmoreexoticantibioticsthatmaybetoxictothecellcultures.Inadditiontheuseofantibioticsmaymaskalowlevelofcontamination. Onceacontaminationhasbeendetected,whetherisitduetobacteria,fungiormycoplasma,therecommendedcourseofactionistodiscardthecultureandcontinuetheworkwithearlierstocksthatareknowntobefreeofcontaminantsorobtainfreshstocksfromarecognizedsource.Howeverifthisisnotpossibleeradicationofthecontaminantmaybeattemptedwiththeuseofantibiotics.InadditionculturecollectionssuchasECACCwillattempttoeradicateanycontaminantsifrequired.PleasecontactECACCforfurtherdetails. Viralinfectionsarevirtuallyimpossibletoremovefromculturessincetheydonotrespondtoantibiotictreatment.Also,sincetheyareintracellularparasitesitisnotpossibletoremovethembycentrifugationorotherseparationtechniques.Ifvirusfreestocksoravirusfreealternativeisnotavailablethenathoroughriskassessmentshouldbeundertakenpriortocontinuingworkwiththeinfectedcellline.