1、ISOTechnicalReportISOTR4752Firstedition2025-06BiotechnologyInventoryofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellcultureBiotechnologieInventairedesmethodespourIadetectiondelacontaminationmicrobiologiquedanslacultureCeIIulairedemammiferesReferencenumberISO/TR4752:2025(en)COPYRIGH
2、TPROTECTEDDOCUMENTISO2025Allrightsreserved.Unlessotherwisespecified,orrequiredinthecontextofitsimplementation,nopartofthispublicationmaybereproducedorutilizedotherwiseinanyformorbyanymeans,electronicormechanical,includingphotocopying,orpostingontheinternetoranintranet,withoutpriorwrittenpermission.P
3、ermissioncanberequestedfromeitherISOattheaddressbeloworISO,smemberbodyinthecountryoftherequester.ISOcopyrightofficeCP401Ch.deBlandonnet8CH-1214Vernier,GenevaPhone:+41227490111Email:copyrightiso.orgWebsite:www.iso.orgPublishedinSwitzerlandContentsPageForewordivIntroductionv1 Scope12 Normativereferenc
4、es13 Termsanddefinitions14 Generalconcepts34.1 Introduction34.2 Detectionprocess35 Contaminationtestinformation46 Criticalcontrolpointsindetection56.1 Samples56.2 ReagentsandEquipment56.3 Operation66.4 Personnel66.5 Environment6Annex A (informative)Availableandexemplarytestingmethodsforbacteriaandfu
5、ngi7Annex B (informative)AvailableandexemplarytestingmethodsforViruses12Annex C (informative)Availableandexemplarytestingmethodsformycoplasma15Bibliography18ForewordISO(theInternationalOrganizationforStandardization)isaworldwidefederationofnationalstandardsbodies(ISOmemberbodies).TheworkofpreparingI
6、nternationalStandardsisnormallycarriedoutthroughISOtechnicalcommittees.Eachmemberbodyinterestedinasubjectforwhichatechnicalcommitteehasbeenestablishedhastherighttoberepresentedonthatcommittee.Internationalorganizations,governmentalandnon-governmental,inliaisonwithISO,alsotakepartinthework.ISOcollabo
7、ratescloselywiththeInternationalElectrotechnicalCommission(IEC)onallmattersofelectrotechnicalstandardization.TheproceduresusedtodevelopthisdocumentandthoseintendedforitsfurthermaintenancearedescribedintheISO/IECDirectives,Part1.Inparticular,thedifferentapprovalcriterianeededforthedifferenttypesofISO
8、documentshouldbenoted.ThisdocumentwasdraftedinaccordancewiththeeditorialrulesoftheISO/IECDirectives,Part2(seeWWW.iso.org/directives).ISOdrawsattentiontothepossibilitythattheimplementationofthisdocumentmayinvolvetheuseof（八）patent(三).ISOtakesnopositionconcerningtheevidence,validityorapplicabilityofany
9、claimedpatentrightsinrespectthereof.Asofthedateofpublicationofthisdocument,ISOhadnotreceivednoticeof（八）patent(三)whichmayberequiredtoimplementthisdocument.HoweverlImplementersarecautionedthatthismaynotrepresentthelatestinformation,whichmaybeobtainedfromthepatentdatabaseavailableatWWW.iso.org/patents.
10、ISOshallnotbeheldresponsibleforidentifyinganyorallsuchpatentrights.Anytradenameusedinthisdocumentisinformationgivenfortheconvenienceofusersanddoesnotconstituteanendorsement.Foranexplanationofthevoluntarynatureofstandards,themeaningofISOspecifictermsandexpressionsrelatedtoconformityassessment,aswella
11、sinformationaboutISOsadherencetotheWorldTradeOrganization(WTO)principlesintheTechnicalBarrierstoTrade(TBT工seeWWW.iso.org/iso/foreword.htmLThedocumentwaspreparedbyTechnicalCommitteeISO/TC276,Biotechnology,SubcommitteeSCLAnalyticalmethods.Anyfeedbackorquestionsonthisdocumentshouldbedirectedtotheusersn
12、ationalstandardsbody.Acompletelistingofthesebodiescanbefoundatwww.iso.org/members.html.IntroductionCellcultureplaysanextremelyimportantroleinbiotechnologyandlifescienceresearch.Therefore,thereliabilityofscientificdatafromcellcultureisessential.Oneofthemostcommonproblemsincellcultureismicrobiological
13、contamination.Thisusuallyresultsincatastrophiccelldeathorcontaminationsustainedatlowlevels(sometimesduetotheuseofantibiotics),andthusimpactsthereliabilityofexperimentaldataderivedfromcellculture.Nonetheless,despiteitsprevalenceandimportance,thelackofopendiscussiondiscouragesdevelopmentandlearningofb
14、estpracticestoavoidmicrobialcontaminationincellculture.Microbialcontaminationisaneconomicissueasitaffectsthereproducibilityofscientificdata,theefficiencyofinvitrocellcultureworkandmostimportantly,thesafetyoflaboratoryoperators.Itisthereforenecessarytomonitormicrobialcontaminationthroughafullundersta
15、ndingofthesourceofcontaminationandemploygoodtestingtechniquesbeforeandduringcellculture.Whenculturedcellshavebeencontaminated,firstthevariousaspectsofthecontaminationareidentified,includingthenatureofcontamination,thetimeofcontaminationandtheoperatingenvironment.Contaminantscanincludebacteria,fungi,
16、mycoplasma,virusesandothertypesoforganisms.Inordertodemonstratetheabsenceofmicrobiologicalcontaminationincellculture,itcanbenecessarytoconductaseriesoftestsforlikelyorganismsandsuchanapproachwillbenefitfromriskassessmentofthecelltype,celloriginandreagentsusedfortheirculture.Environmentalmonitoringin
17、areaswherecellsareculturedandstoredcanreducetheriskofmicrobialcontaminationfromtheprocessingenvironmentThisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellcultureinordertoprovideuserswithanoverviewthatalsoincludesinformationonpro-andcontraindicationsfo
18、rthelistedmethodsinrelationtotheappropriatesampletype.BiotechnologyInventoryofmethodsfordetectionofmicrobiologicalcontaminationinmammaliancellculture1 ScopeThisdocumentprovidesaninventoryofmethodsforthedetectionofmicrobiologicalcontaminationinmammaliancellculture.Thisdocumentincludesconsiderationsfo
19、rtheselectionofmethodstotestthepresenceofcommoncontaminantssuchasbacteria,fungi,virusesandmycoplasma.Thisdocumentisnotapplicabletoprionsandprotists.Thisdocumentisintendedforusebybiomedicalresearchers,biobankoperatorsandothersperformingmammaliancellculture.2 NormativereferencesTherearenonormativerefe
20、rencesinthisdocument.3 TermsanddefinitionsForthepurposesofthisdocument,thefollowingtermsanddefinitionsapply.ISOandIECmaintainterminologydatabasesforuseinstandardizationatthefollowingaddresses: ISOOnlinebrowsingplatform:availableathttps:WWW.iso.org/obp IECElectropedia:availableathttps:WWW.electropedi
21、a.org/1.1accuracyclosenessofagreementbetweenameasuredquantityvalueandthetruequantityvalueofameasurandSOURCE:ISO/IECGuide99:2007,2.13,modifiedNotestoentrydeleted.1.2digitalPCRdPCRprocedureinwhichnucleicacidtemplatesaredistributedacrossmultiplepartitionsofnominallyequivalentvolume,suchthatsomepartitio
22、nscontaintemplateandothersdonot,followedbyPCRamplificationoftargetsequencesanddetectionofspecificPCRproducts,providingacountofthenumberofpartitionswithapositiveandnegativesignalforthetargettemplateNote1toentry:Nucleicacidtargetsequencesareassumedtoberandomlyandindependentlydistributedacrossthepartit
23、ionsduringthepartitioningprocess.Note2toentry:ThecountofpositiveandnegativepartitionsisnormallybasedonendpointdetectionofPCRproductsfollowingthermalcycling,howeverreal-timeqPCRmonitoringofPCRproductaccumulationisadditionallypossibleforsomedPCRplatforms.SOURCE:ISO20395:2019,3.101.3intendeduseintended
24、purposeuseforwhichaproduct,process,orserviceisintendedaccordingtothespecifications,instructionsorinformationormultipleofthemprovidedbythemanufactureroruserSOURCE:ISO23033:2021,3.261.4microbialcontaminationpresenceofunintendedbacteria,fungi,mycoplasma,orvirusesSOURCE:ISO11139:2018,3.1711.5nextgenerat
25、ionsequencingNGSnon-Sanger-basedhigh-throughputnucleicacidsequencingNote1toentry:Millionsorbillionsofnucleicacidstrandscanbesequencedinparallel,yieldingsubstantiallymorethroughput.Note2toentry:NGS(next-generation-sequencing)isalsowellrecognizedasMPS(massivelyparallelsequencing)intheISO20397series.No
26、te3toentry:MPSorNGScoverslongreadsequencingandshortreadsequencing.SOURCE:ISO/DIS20397-3:2024,3.161.6PCRassayqPCRordPCR(3.2)measurementmethodwithspecifiedoligonucleotideprimers(and,insomecases,aprobeorprobes)thatisusedtoidentifyorquantifyanucleicacidtargetSOURCE:ISO20395:2019,3.231.7reagentsubstanceu
27、sedinchemical/biochemicalanalysisorotherreactionSOURCE:ISO20391-1:2018,3.191.8resolutionsmallestchangeinaquantitybeingmeasuredthatcausesaperceptiblechangeinthecorrespondingindicationNote1toentry:Resolutioncandependon,forexample,ratioofsignalandnoise(internalorexternal.Itcanalsodependonthevalueofaqua
28、ntitybeingmeasured.SOURCE:JCGM200:2012,4.14,modifiedFrictionnotmentionedinNote1toentry.1.9ribosomalRNArRNAnon-codingribonucleicacidcontainedinribosomesNoteltoentryiThenucleotidesequenceofrRNAsubunitscanbeusedtodetectmicroorganisms(e.g.16SrRNA,18SrRNA.3.10sampleoneormorepartstakenfromasystemSOURCE:IS
29、O23033:2021,3.463.11sensitivityquotientofthechangeinanindicationofameasuringsystemandthecorrespondingchangeinavalueofaquantitybeingmeasuredNote1toentry:Sensitivityofameasuringsystemcandependonthevalueofthequantitybeingmeasured.Note2toentry:Thechangeconsideredinavalueofaquantitybeingmeasuredmustbelar
30、gecomparedwiththeresolution.SOURCE:ISO/IECGuide99:2007,4.12,modifiedsensitivityisgivenastheonlypreferredterm.3.12testsamplesmallaliquotofthesamplethatispreparedformeasurementinthemethodofinterestNote1toentry:Generally,testsamplesarerepresentativeofthesampletheyarepreparedfromandaresometimesreferredt
31、oasrepresentativetestSamPle(三)”.SOURCE:ISO20391-2:2019,3.363.13validationconfirmation,throughtheprovisionofobjectiveevidence,thattherequirementsforaspecificintendeduseorapplicationhavebeenfulfilledSOURCE:ISO23033:202L3.544 Generalconcepts4.1 IntroductionContaminationofcellculturecanproduceadverseeff
32、ectsincellphenotypeandquality,aswellasapplicationsinresearchanddevelopmentincludingclinicalstudies,stemcellmanufacturingetc.Diversemethodsandtechniqueshavebeendevelopedforlaboratorymonitoringandtestingofmicrobialcontaminants.Manymicrobialcontaminantsespeciallycoarsecontaminationbybacteriaandfungican
33、leadtosignificantabnormalitiesincellviabilityandappearance,whichcanbevisuallyobservedbythenakedeyeorunderamicroscope.However,manycontaminantssuchaslowlevelsofbacteriaandfungi,mycoplasmaandvirusescannotbedetectedbychangesincellappearanceandcellculturemediumthroughdirectobservation.Thus,appropriatetec
34、hniquesareusedfortestingmicrobialcontaminantsbasedonthecharacteristicsofthemicrobialcontaminantsandlaboratoryfacilities.4.2 DetectionprocessThedetectionofmicrobiologicalcontaminationinmammaliancellculturesiscarriedoutunderasepticconditions.NOTE1Airqualityofthetestingenvironmentcanbedefinedaccordingt
35、oISOstandardsforcertainregulatedapplications.FurtherguidanceisgiveninISO16000-44andISO1105733.Thedetectionofmicrobiologicalcontaminationofmammaliancellsincludingbacteria,fungi,viruses,andmycoplasmadoesnotspecificallyconsidertheevaluationofthelikelihoodofcontaminationbyotherorganismgroupslikeprionsan
36、dprotists.NOTE2Mycoplasmaisabacterium(orgerm)thatcaninfectcellculturesanddifferentpartsofhumanbody.Unlikeotherbacteria,mycoplasmadonothavecellwalls.Theyarealsoverysmallcomparedtootherbacteria.Manyantibioticskillbacteriabyweakeningthosewalls.Sincemycoplasmabacteriadonthavethem,someantibiotics,likepen
37、icillin,wontworkagainstthem.Mycoplasmaisdiscussedseparatelyfrombacteriainthisdocument.Ariskassessmenttoidentifyanylikelycontaminantscanfacilitateregimeestablishmentformicrobiologicalcontaminationdetectioninaparticularcellculture.Thisregimehelpstoaddressanypotentialmicrobiologicalhazardsassociatedwit
38、hthederivation,culturemethods,mediaandstoragehistoryofthecellculturetobetested.Thetestingofcellculturesforbacteria,fungi,viruses,mycoplasmaisusuallyconsideredtobearoutineprocedureforanymammaliancellcultures,unlessspecificconditionsapplytorenderthisunnecessary.Certainorganismsthatarecommoncontaminant
39、sarisingfromthegenerallaboratoryenvironmentandothertypecellculturescanbeconsideredinmicrobialdetectionforanymammaliancellculture.Considerationsforselectionofthetestmeasurementsdependontheintendedpurposeaswellassampleandprocessingfactorsthatintroduceadditionalpotentialformicrobialcontamination.Thesei
40、nclude,butarenotlimitedto,theintendedpurposeforcells,celltypes,cellattributes,potentialeffectsofsampleheterogeneity,andthepresenceofinhibitorysubstancesinsamplestobeusedforthedetection.Thelaboratoryestablishesproceduresforthepreparationofcontrolsandreferencematerials,including,butnotlimitedto,calibr
41、ation,validation,expirydateandhazardidentification.Detailsofrawdata,controlsandresultsarenormallyrecordedanddocumentedforthepurposeofqualitycontrol,review,auditandassessment.NOTE3Suchanapproachisincapableofpredictinganddetectingallformsofcontamination(ie,itcannotassertthatacultureisfreeofcontaminati
42、on).5 ContaminationtestinformationTheusersfirstmakeriskassessmenttodeterminethepossiblecontaminationbeforemicrobialcontaminationtesting.Thisassessmentdependsonthesourceofcells,thehistoryofexposuretopotentialcontamination,theexpectedapplicationpurposeofcellsandtheenvironment.Cellsfromdifferentspecies
43、arelikelytocarrydifferentpotentialmicroorganisms.Theexposurehistoryofthecellcultureistakenintoaccountincludingadetailedassessmentofrawmaterialsandothermaterialswhichcanhavecomeintodirectcontactwiththecellsinquestion.Rawmaterialsofbiologicaloriginareofspecialconcernsincetheycantransmitorganismspresen
44、tintheoriginalsourcematerials.Inparticular,materialsderivedfromhigherriskmammaliantissuesandbodyfluidsareassessedforriskofinfectionofthefinalmaterialsdependingonthelikelihoodofcontaminationanditsinactivationorremovalduringprocessing.Ofspecialconcernareanimalserumandotherrawmaterialsusedwidelytocultu
45、recellsareofspecialconcernastheycanbecomecontaminatedbyvirusespresentindonorswhichcannotbesubjectedtoeffectivedisinfectionorsterilization.Relevantcontaminationdetectioncanbecarriedoutaccordingtothepurposeofcelluse,withrespecttothesafetyoflaboratorypersonnel,thetypeandqualityofdataobtainedfromcellcul
46、ture-basedassays,andthepotentialforongoingapplicationofthetestedcellculturewhereanycontaminantscanbepassedtoothercultures,e.g.asfeedercells,toprovideconditionedcellculturemedia,orextracellularmatrix.Theenvironmentalfactorsofcellpreparationandcellculturecancausecontamination.Possiblecontaminationcanb
47、eassessedbasedonexposurehistory,e.g.animalserumusedtoculturecellscanbecontaminatedbyspecificmicroorganisms.Understandingtheexistingdetectionmethods,theircharacteristics,andtheirdetectioncapabilitiesisimportantinordertomeettheneededdetectionrequirements.Inthecontextoftheriskassessment,theanalyticalla
48、boratoryevaluatestheapplicabilityofexistingtestmethods,andselectstheappropriatemethodforthesamplestobetested.DetailsarelistedinAnnexA,AnneXBandAnneXCforeachtypeofmethod.Fullconsiderationofthetestingapproachincludestypesofcontamination(e.g.specificmicroorganismsorbroadercontaminants),andtestresolution(e.g.llimitsofdetectionandlimitsofquantification)andspecificityoftechniques.Selectionofanappropriatetestingmethodconsidersfactorsincludingtargetmicroorganisms,characteristicsofsamples,andhandli
