研究用肿瘤细胞系的质量控制和要求课件

研究用肿瘤细胞系的质量

控制和要求

中国医学科学院基础医学研究所刘玉琴研究用肿瘤细胞系的质量控制和要求SomeFactsabouttheuseofculturedcells--Nobelprize2001，Cellcycle2002,apoptosis2006,RNAInterference2007,homologousrecombinationandgenetargeting2008,HPV—cervicalca,HIV--aids2009,telomere&telomeraseNobelprizewinner,useinvitroculturedcellsfortheirresearches.研究用肿瘤细胞系的质量控制和要求现代生命科学研究中培养细胞的使用越来越广泛1969~2004：celluseincreasingsteadilyPapersusecellsincrease2~2.5Xbiology,pharmacologyandmedicine----tumorresearch不可少的研究材料、对象、工具Useofcontaminatedormisidentifiedcellsincrease10X研究用肿瘤细胞系的质量控制和要求Commonproblemsmicrobiologicalcontaminationwithparticularrespecttomycoplasmainfections(incidenceofca.25%)cross-contaminationbyothercelllinesandmisidentificationormisclassification(incidenceofmorethan15%)acquisitionofgeneticandepigeneticvariationsduetoover-passagingofcelllinesleadingtoageneticdriftofcellculturesandtotheselectionofalteredsubclones.研究用肿瘤细胞系的质量控制和要求Wherefrom?faultycellculturetechniques:sharedreagents,repeateduseofthesamepipetteduringre-feedingoperationsandmanipulationofmultipleculturesatthesametimewithoutadequateisolationofonecelltypefromanother.theintroductionoffalse,contaminatedorover-passagedcelllinesfromothersourcesintothecellculturelaboratory(35%)Mislabeling,lackofawarenessIntentionalco-cultivationduringpropagationofhumanstemorprimarycellsusingafeederlayerderivedfromanotherspecies研究用肿瘤细胞系的质量控制和要求细胞交叉污染历史回顾1968,ApparentHeLacellcontaminationofhumanheteroploidcelllines.Nature.217:750-7511974，HeLacesscontaminatingculturesaroundtheworld,Sciences184:10591981,Contaminatedcellcultures.Nature289:2271981,cross-contaminationofcellsinculture.Sciences.212(4493):226-52研究用肿瘤细胞系的质量控制和要求细胞交叉污染历史回顾1999,Falsehumanhematopoieticcelllins:cross-cantaminationsandmisinterpretations.Leukemia.13(10):1601-72003,Falseleukmia–lymphomacelllines:anupdateonover500celllines.Leukemia17,416-4262004,celllinecross-comtamination:howawareare…invitrocelldevbiolanim.40(7):211-52007,Casesofmistakenidentity.Sciences315:928-9312009,NaturecallsforglobaldatabaseofcelllineSTRprofiles研究用肿瘤细胞系的质量控制和要求保藏机构公布的错误细胞1细胞系种属细胞类型污染源种属细胞类型数据来源207人前B白血病REH人前B白血病DSMZ2474/90人胃癌HT-29人结直肠癌DSMZ2957/90人胃癌HT-29人结直肠癌DSMZ3051/80人胃癌HT-29人结直肠癌DSMZADLC-5M人肺癌HELA/-S3人宫颈癌DSMZAV3人羊膜HeLa人宫颈癌ATCCBCC-1/KMC人基底细胞癌HELA/-S3人宫颈癌DSMZBM-1640人前列腺癌DU-145人前列腺癌DSMZC16人胚胎肺成纤维细胞（MRC-5克隆）HeLa人宫颈癌ECACC研究用肿瘤细胞系的质量控制和要求保藏机构公布的错误细胞2CHANGLiver人胚胎肝上皮HeLa人宫颈癌ATCC;JCRBCOLO-818人黑色素瘤COLO800人黑色素瘤DSMZDAMI人巨核细胞HEL人红系白血病DSMZECV304人内皮T24人膀胱癌ATCCECV-304人正常内皮T24人膀胱癌DSMZEJ人膀胱癌T24人膀胱癌ATCC;JCRBEPLC3-2M1人肺癌HELA/-S3人宫颈癌DSMZ;JCRBEPLC-65人肺癌HELA/-S3人宫颈癌DSMZF2-4E5人胸腺上皮SK-HEP-1人肝癌DSMZF2-5B6人胸腺上皮SH-HEP-1人肝癌DSMZFL人羊膜HeLa人宫颈癌ATCCGHE人星状细胞瘤T-24人膀胱癌DSMZGirardiHeart猪成年心脏HeLa人宫颈癌ATCC研究用肿瘤细胞系的质量控制和要求HAG人T-24人膀胱癌DSMZHEp-2人成年喉上皮HeLa人宫颈癌ATCCHMV-1人黑色素瘤HeLa-S3人宫颈癌DSMZHuL-1HeLa人宫颈癌JCRBIMC-2人颚骨癌HeLa-S3人宫颈癌DSMZIntestine407人小肠上皮HeLa人宫颈癌ATCCJ-111HeLa人宫颈癌JCRBJOSK-I人单核细胞白血病U-937人组织细胞淋巴瘤DSMZJOSK-K人单核细胞白血病U-937人组织细胞淋巴瘤DSMZJOSK-M人单核细胞白血病U-937人组织细胞淋巴瘤DSMZ保藏机构公布的错误细胞3研究用肿瘤细胞系的质量控制和要求JOSK-S人单核细胞白血病U-937人组织细胞淋巴瘤DSMZJTC-17

HeLa人宫颈癌Yamakage(JCRB)KB人HeLa人宫颈癌ATCC;JCRBKO51K562人髓系白血病DSMZ;JCRBKOSC-3Ca9-22人JCRBL132人胚胎肺上皮HeLa人宫颈癌ATCCLR10.6人前B白血病NALM-6人前B白血病DSMZMaTu人乳腺癌HELA/-S3人宫颈癌DSMZMC-4000人乳腺癌HELA/-S3人宫颈癌DSMZMKB-1人T细胞白血病CCRF-CEM人T细胞白血病DSMZ保藏机构公布的错误细胞4研究用肿瘤细胞系的质量控制和要求MKN28MKN74人JCRBMOLT-15人T细胞白血病CTV-1人单核细胞白血病DSMZMT-1人乳腺癌HELA/-S3人宫颈癌DSMZNCC16PHK16-0b人JCRBP1-1A3人胸腺上皮SK-HEP-1人肝癌DSMZP1-4D6人胸腺上皮SK-HEP-1人肝癌DSMZP39TSU人HL60人髓系白血病JCRBPBEI人前B白血病NALM-6人前B白血病DSMZPSV811人成纤维WI38人成纤维JCRBRAMAK-1人肌肉滑膜T-24人膀胱癌DSMZ保藏机构公布的错误细胞5研究用肿瘤细胞系的质量控制和要求SBC-2人膀胱癌HELA/-S3人宫颈癌DSMZSBC-7人膀胱癌HELA/-S3人宫颈癌DSMZSCLC-16H人SCLCSCLC-21/22H人SCLCDSMZSCLC-24H人SCLCSCLC-21/22H人SCLCDSMZSNB-19U251MG人胶质瘤ATCCSPI-801人T细胞白血病K562人髓系白血病DSMZSPI-802人T细胞白血病K562人髓系白血病DSMZTK-1人?U251MG人胶质瘤JCRBTMH-1IHH-4人JCRBWISH人新生羊膜上皮HeLa人宫颈癌ATCC保藏机构公布的错误细胞6研究用肿瘤细胞系的质量控制和要求DSMZ德国细胞库550leukemiaandlymphomacelllinessubmitted59/395(15%)byoriginators23 /155(15%)bysecondarysources研究用肿瘤细胞系的质量控制和要求交叉污染细胞使用的后果Erroneousresults:WastedresearchmoneyPotentialdelayindiscovery研究用肿瘤细胞系的质量控制和要求错误细胞使用2000-2004：19/Int407;45/WISH;59/Changliver;470/Hep-2;556/KB;2004(Buehring),pubmedsearch220publicationsinvolved,32%useHeLacells,33%testedforauthenticity,35%getcellsfromotherlabs.NCIpanelof60celllineswereusedforanticancerdrugevaluation.AmongthemADR-RESisacutallyOVCAR-8.Ithadbeenusedinabout300papers.研究用肿瘤细胞系的质量控制和要求HeLacervicalcancercellsInt-407(describedas“non-transformedintestinalepithelialcells”)inBr.J.Cancer101,1596(2009),EMBOJ.22,5003(2003)andJ.Biol.Chem.280,13538(2005)WISH(describedas“non-transformedamnioticepithelialcells”)inMol.Pharmacol.69,796(2006),Endocrinology147,2490(2006)andJ.Biol.Chem.278,31731(2003)Changliver(describedas“normallivercells”)inOncogene28,3526(2009),Proteomics14,2885(2008)andJ.Biol.Chem.279,28106(2000)HEp-2(describedas“laryngealcancer”)inInvestig.NewDrugs26,111–118(2008),Carcinogenesis29,1519(2008)andJ.Biol.Chem.283,36272(2008)KB(describedas“oralcancer”)inBiochem.Pharmacol.73,1901–1909(2007),Clin.CancerRes.14,8161(2008)andJ.Biol.Chem.280,23829(2005)HeLa,Int-407andHEp-2cellswereusedasthreedistinctcelllinesinthesamestudyinCancerRes.69,632(2009)研究用肿瘤细胞系的质量控制和要求Ecv-304,“人脐静脉内皮细胞”日本实验室建立，ATCC、DSMZ等机构有保藏，在1999—2000年，经STR检测，证明为T24(膀胱癌)，各自网站有公布。在实验室间广泛传播，2006y,全世界600余论文，利用该细胞进行研究，2/3在中国。Why?Donotknow?NoothersuitablecellularmodelExpensieve,cannotafford,havetocompromise研究用肿瘤细胞系的质量控制和要求Ecv-304/T24研究用肿瘤细胞系的质量控制和要求对细胞质量的要求2007.10,Goslar,Germany，11ICCCScientistfromATCCproposed:(1)fundingagencies,gov.orprivaterequirecelllineauthenticationasaconditionfortheawardofgrantandcontractsfunds.(2)keyscientificJ…asaconditionforpublication.(3)conference,workshopand/ortraining.(4)labheadsbeencouragedtoensureQCmeasures.Authentic,WellcharacterizedTheproblemofSub-culturing研究用肿瘤细胞系的质量控制和要求NIHNOTICE2007.12,RequirecellanthenticationForgrantForpaper研究用肿瘤细胞系的质量控制和要求杂志及细胞使用者的要求

Examples1Thp-1,2009-1-5celllineauthentication,characterizationandfreedomfromcontaminationThisisanimportantissue,towhichauthorsshouldpayparticularattention.Itistheauthors'responsibilitytoensurethatcelllinesusedinanystudy(andthemajoritydoinvolvethem)arecertifiedasbeingthedesignatedtype,thattheyhavebeencheckedtoensuretheyarefreeofcontamination(e.g.mycoplasma),andthattheyhavebeenusedfromyoungstock.Thesearethethreecriteriauponwhichgoodpracticeisbased,assetoutintheUKCCCGuidelinesontheuseofcelllines().AttentionshouldbepaidtothisintheMaterialandMethodssection,andauthorsomittingtoincludeevidenceofgoodpracticewillfindthattheirarticlesmaybedelayeduntilsuchguaranteesaregiven.研究用肿瘤细胞系的质量控制和要求Example2,ReportingonCellLineUseinAACRJournalsNEW!EFFECTIVEJULY1,2009

AACRstronglyencouragestheauthenticationofcelllinesusedintheresearchreportedinitsjournals.Ifcelllineswereusedintheresearch,astatementaddressingthefollowingpointsmustbeincludedinthe"MaterialsandMethods"section:1.

Fromwhereandwhenthecellswereobtained2.

Whetherthecelllineshavebeentestedandauthenticated3.

Themethodbywhichthecellsweretested4.

Howandwhenthecellswerelasttested研究用肿瘤细胞系的质量控制和要求AACRJournalsCancerResearchClinicalCancerResearchCancerEpidemiology,Biomarkers&Prevention，

CEBPCancerPreventionResearchMolecularCancerResearchMolecularCancerTherapeuticsCancerReviewsOnlinePreventionPortal研究用肿瘤细胞系的质量控制和要求Example2—shelflifeIfcellswereobtaineddirectlyfromacellbankthatperformscelllinecharacterizationsandpassagedintheuser'slaboratoryforfewerthan6

monthsafterreceiptorresuscitation,re-authorizationisnotrequired.Inthesecases,pleaseincludethemethodofcharacterizationusedbythecellbank.Ifcellswereobtaineddirectlyfromacellbankthatperformscelllinecharacterizationsandpassagedintheuser'slaboratoryforfewerthan6

monthsafterreceiptorresuscitation,re-authorizationisnotrequired.Inthesecases,pleaseincludethemethodofcharacterizationusedbythecellbank.研究用肿瘤细胞系的质量控制和要求KeymilestonesintheefforttoaddresscelllinemisidentificationHelacelllineestablishedfromHenriettaLacksATCC,AmericanTypeCultureCollection.NIHNationalInstitutesofHealth.STR,ShortTandemRepeatInterspeciescross-ContaminationshownATCCstartsCuratingcelllinesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesNelson-Rossconfirms&estendsStanGartler’sfindingsJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantes1981-2005crossContaminationspreedsBeyongdHeLacellsNIHterminatescontractofNelson-RossNIHissuesguidelinesToavoidtheuseofmisidentifiedcelllinesRolandNardonestartstheSecondcrusadeagsinstCorsscontaminationNaturecallsforaGlobaldatabaseofCelllinesSTRprofies1952195819621966197419801981200520072009HelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllines1981-2005crossContaminationspreedsBeyongdHeLacellsNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesNIHissuesguidelinesToavoidtheuseofmisidentifiedcelllines1981-2005crossContaminationspreedsBeyongdHeLacellsNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllines1952195819621966197419801981200520072009Interspeciescross-Contaminationshown1952195819621966197419801981200520072009StanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009StanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009JohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009JohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009NIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009NIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009RolandNardonestartstheSecondcrusadeagsinstCorsscontaminationNIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009RolandNardonestartstheSecondcrusadeagsinstCorsscontaminationNIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009NaturecallsforaGlobaldatabaseofCelllinesSTRprofiesRolandNardonestartstheSecondcrusadeagsinstCorsscontaminationNIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981200520072009NIHissuesguidelinesToavoidtheuseofmisidentifiedcelllines1981-2005

crossContaminationspreedsBeyongdHeLacellsNelson-Rossconfirms&estendsStanGartler’sfindingsHelacelllineestablishedfromHenriettaLacksATCCstartsCuratingcelllinesNaturecallsforaGlobaldatabaseofCelllinesSTRprofiesRolandNardonestartstheSecondcrusadeagsinstCorsscontaminationNIHterminatescontractofNelson-RossJohnMaddoxdescribesIndividuallikeNelson-RossAsself-appointedvigilantesStanGartlershowsintraspeciesCross-contaminationbetweenHumancelllinesInterspeciescross-Contaminationshown1952195819621966197419801981