Introduction

Qualityisimportantinallaspectsoftissueculturesincethequalityofmaterialsusedi.e.mediaandotherreagents)willaffectthequalityoftheculturesandproductsderivedfromthem.Themainareasofqualitycontrolthatareofconcernfortissuecultureare:

• Thequalityofthereagentsandmaterials
• Theprovenanceandintegrityofthecelllines
• Theavoidanceofmicrobialcontamination

ReagentsandMaterials

Apotentialsourceofcontaminationisreagentsandmaterials,inparticularbovineserumwhichhasbeenidentifiedasasourceofbovineviraldiarrhoeavirus(BVDV).PorcinetrypsinisalsoapotentialsourceofMycoplasmahyorhinis.Goodqualityreagentsandmaterialsareavailablefromnumerousmanufacturersoftissueculturemediaandsupplements.InadditionmanufacturersincludingSigmawillcarryoutarangeofqualitycontroltestsincludingscreeningformycoplasmaandBVDVandsupplyaCertificateofAnalysiswiththeirproducts.Thesestatetheproductandlotnumbersandformsavitalpartofrecordkeepingandtrackingofreagentsusedintheproductionofcellstocks.ItisadvisabletofurthertestkeyreagentssuchasFBStoensurethattheyare‘fitforpurpose’duetobatch-to-batchvariation.

Manufacturersofsterileplasticware(flasks,centrifugetubes,Pipettes)designedfortissuecultureusearealsosuppliedwithCertificatesofAnalysisforeachbatchproduced,whichshouldbekeptforfuturereference.

ProvenanceandIntegrityofCellLines

Thesourcingofcelllinescanhaveanimportanteffectonqualitysincefreshlyimportedcelllinesareamajorsourceofcontamination.Theadvantagesofobtainingcelllinesfromarecognizedsourcesuchasaculturecollectionare:

• Contaminantfree
• FullycharacterizedandauthenticatedintermsofDNAprofileandspeciesoforigin
• Suppliedwithadetaileddatasheet

Oncecelllineshavebeenobtainedfromareputablesourceitisimportanttoimplementmasterandworkingcellbankingproceduresandtheassociatedqualitycontrolstepssuchasroutinetestingformicrobialcontaminantsandconfirmingtheidentityofcultures.

AvoidanceofMicrobialContamination

Potentialsourcesofcontaminationincludeothercelllines,laboratoryconditionsandstaffpoorlytrainedincoreareassuchasaseptictechniquesandgoodlaboratorypractice.Thustheuseofcellsandreagentsofknownoriginandqualityaloneisnotsufficienttoguaranteequalityofproduct(cellstockorcultureproducts);itisnecessarytodemonstratequalitythroughouttheproductionprocessandalsointhefinalproduct.Routinescreeningaidstheearlydetectionofcontaminationsinceallmanipulationsareapotentialsourceofcontamination.

The3maintypesofmicrobialcontaminantsintissuecultureare:

• BacteriaandFungi
• Mycoplasma
• Viruses

BacterialandFungalContaminationBacterialcontaminationisgenerallyvisIBLetothenakedeyeanddetectedbyasuddenincreaseinturbidityandcolorchangeoftheculturemediumastheresultofachangeinpH.Thecellculturemaysurviveforashorttimebutthecellswilleventuallydie.Dailymicroscopicobservationofcultureswillensureearlydetectionofcontaminationandenableappropriateactiontobetakenassoonasthefirstsignsofcontaminationbecomeapparent(seebelow).Inadditionspecifictestsforthedetectionofbacteriaandfungishouldbeusedaspartofaroutineandregularqualitycontrolscreeningprocedure(seeProtocol8).

MycoplasmaContaminationMycoplasmasarethesmallestfree-livingself-replicatingprokaryotes.TheylackacellwallandlacktheABIlitytosynthesizeone.Theyare0.35mindiameterandcanbeobservedasfilamentousorcoccalforms.Thereare5majorspeciesthataretissueculturecontaminants,namelyM.hyorhinis,M.arginini,M.orale,M.fermentansandAcholeplasmalaidlawii.

Theeffectsofmycoplasmainfectionaremoreinsidiousthanthoseofbacteriaandfungiinducingseverallongtermeffects.Theseinclude:

• Reducedgrowthrate
• Morphologicalchanges
• Chromosomeaberrations
• Alterationsinaminoacidandnucleicacidmetabolism

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.