研究用肿瘤细胞系的质量控制和要求课件.ppt

1、v2001，Cell cyclev2002,apoptosisv2006,RNA Interferencev2007,homologous recombination and gene targetingv2008,HPVcervical ca,HIV-aidsv2009,telomere&telomeraseNobel prize winner,use in vitro cultured cells for their researches.v19692004：cell use increasing steadilyvPapers use cells increase 22.5Xv biol

2、ogy,pharmacology and medicine-tumor researchv不可少的研究材料、对象、工具vUse of contaminated or misidentified cells increase 10Xvmicrobiological contamination with particular respect to mycoplasma infections(incidence of ca.25%)vcross-contamination by other cell lines and misidentification or misclassification(i

3、ncidence of more than 15%)vacquisition of genetic and epigenetic variations due to over-passaging of cell lines leading to a genetic drift of cell cultures and to the selection of altered subclones.v faulty cell culture techniques:shared reagents,repeated use of the same pipette during re-feeding op

4、erations and manipulation of multiple cultures at the same time without adequate isolation of one cell type from another.v the introduction of false,contaminated or over-passaged cell lines from other sources into the cell culture laboratory(35%)vMislabeling,lack of awarenessvIntentional co-cultivat

5、ion during propagation of human stem or primary cells using a feeder layer derived from another speciesv1968,Apparent HeLa cell contamination of human heteroploid cell lines.Nature.217:750-751v1974，HeLa cess contaminating cultures around the world,Sciences 184:1059v1981,Contaminated cell cultures.Na

6、ture 289:227v1981,cross-contamination of cells in culture.Sciences.212(4493):226-52v1999,False human hematopoietic cell lins:cross-cantaminations and misint erpretations.Leukemia.13(10):1601-7v2003,False leukmia lymphoma cell lines:an update on over 500 cell lines.Leukemia 17,416-426v2004,cell line

7、cross-comtamination:how aware arein vitro cell dev biol anim.40(7):211-5v2007,Cases of mistaken identity.Sciences 315:928-931v2009,Nature calls for global database of cell line STR profiles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

8、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ECACCCHANG Liver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

9、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DSMZGirardi Heart猪成年心脏HeLa人宫颈癌ATCCHAG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DSMZIntestine 407人小肠上皮HeLa人宫

10、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DSMZJOSK-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/

11、-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DSMZMKN28MKN74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DSMZP39 TSU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D

12、SMZSBC-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ATCCv550 leukemia and lymphoma cell lines submitted

13、v59/395(15%)by originatorsv23/155(15%)by secondary sourcesvErroneous results:vWasted research moneyvPotential delay in discoveryv2000-2004：19/Int 407;45/WISH;59/Chang liver;470/Hep-2;556/KB;v2004(Buehring),pubmed search 220publications involved,32%use HeLa cells,33%tested for authenticity,35%get cel

14、ls from other labs.vNCI panel of 60 cell lines were used for anticancer drug evaluation.Among them ADR-RES is acutally OVCAR-8.It had been used in about 300 papers.v Int-407(described as“non-transformed intestinal epithelial cells”)in Br.J.Cancer 101,1596(2009),EMBO J.22,5003(2003)and J.Biol.Chem.28

15、0,13538(2005)v WISH(described as“non-transformed amniotic epithelial cells”)in Mol.Pharmacol.69,796(2006),Endocrinology 147,2490(2006)and J.Biol.Chem.278,31731(2003)v Chang liver(described as“normal liver cells”)in Oncogene 28,3526(2009),Proteomics 14,2885(2008)and J.Biol.Chem.279,28106(2000)v HEp-2

16、(described as“laryngeal cancer”)in Investig.New Drugs 26,111118(2008),Carcinogenesis 29,1519(2008)and J.Biol.Chem.283,36272(2008)vKB(described as“oral cancer”)in Biochem.Pharmacol.73,19011909(2007),Clin.Cancer Res.14,8161(2008)and J.Biol.Chem.280,23829(2005)vHeLa,Int-407 and HEp-2 cells were used as

17、 three distinct cell lines in the same study in CancerRes.69,632(2009)v日本实验室建立，ATCC、DSMZ等机构有保藏，在19992000年，经STR检测，证明为T24(膀胱癌)，各自网站有公布。v在实验室间广泛传播，v2006y,全世界600余论文，利用该细胞进行研究，2/3在中国。Why?vDonot know?vNo other suitable cellular modelvExpensieve,can not afford,have to compromisev2007.10,Goslar,Germany，11IC

18、CCvScientist from ATCC proposed:(1)funding agencies,gov.or private require cell line authentication as a condition for the award of grant and contracts funds.(2)key scientific J as a condition for publication.(3)conference,workshop and/or training.(4)lab heads be encouraged to ensure QC measures.v A

19、uthentic,Well characterizedvThe problem of Sub-culturingv2007.12,vRequire cell anthenticationvFor grantvFor papervThp-1,2009-1-5vcell line authentication,characterization and freedom from contamination vThis is an important issue,to which authors should pay particular attention.It is the authors res

20、ponsibility to ensure that cell lines used in any study(and the majority do involve them)are certified as being the designated type,that they have been checked to ensure they are free of contamination(e.g.mycoplasma),and that they have been used from young stock.These are the three criteria upon whi

21、ch good practice is based,as set out in the UKCCC Guidelines on the use of cell lines(http:/ukcccr.icnet.uk).Attention should be paid to this in the Material and Methods section,and authors omitting to include evidence of good practice will find that their articles may be delayed until such guarante

22、es are given.vNEW!EFFECTIVE JULY 1,2009 vAACR strongly encourages the authentication of cell lines used in the research reported in its journals.If cell lines were used in the research,a statement addressing the following points must be included in the Materials and Methods section:1.From where and

23、when the cells were obtained 2.Whether the cell lines have been tested and authenticated 3.The method by which the cells were tested 4.How and when the cells were last tested vCancer Research vClinical Cancer Research vCancer Epidemiology,Biomarkers&Prevention，CEBP vCancer Prevention Research vMolec

24、ular Cancer Research vMolecular Cancer Therapeutics vCancer Reviews Online vPrevention Portal vIf cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6 months after receipt or resuscitation,re-authorization is no

25、t required.In these cases,please include the method of characterization used by the cell bank.vIf cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6 months after receipt or resuscitation,re-authorization is no

26、t required.In these cases,please include the method of characterization used by the cell bank.Hela cell line establishedfrom Henrietta LacksATCC,American Type Culture Collection.NIH National Institutes of Health.STR,Short Tandem RepeatInterspecies cross-ContaminationshownATCC starts Curating cell li

27、nesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesNelson-Ross confirms&estends Stan Gartlers findingsJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes1981-2005 crossContamination spreeds Beyongd HeLa cellsNIH terminatescontract of Nelson-RossNIH

28、issues guidelinesTo avoid the use of misidentified cell linesRoland Nardone starts the Second crusade agsinst Corss contaminationNature calls for aGlobal database ofCell lines STR profies1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Hela cell line establishedfrom Henrietta LacksATCC starts Curat

29、ing cell linesHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNelson-Ross confirms&estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNels

30、on-Ross confirms&estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNelson-Ross confirms&estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell lines1981-2005 crossContamination spreeds Beyongd HeL

31、a cellsNelson-Ross confirms&estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNIH issues guidelinesTo avoid the use of misidentified cell lines1981-2005 crossContamination spreeds Beyongd HeLa cellsNelson-Ross confirms&estends Stan Gartlers fi

32、ndingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell lines1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Interspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Stan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInters

33、pecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Stan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 John Maddox describesIndividual like Nelson-RossAs self-ap

34、pointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamin

35、ation betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 NIH terminatescontract of Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell li

36、nesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 NIH terminatescontract of Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Cont

37、aminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Roland Nardone starts the Second crusade agsinst Corss contaminationNIH terminatescontract of Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betw

38、eenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Roland Nardone starts the Second crusade agsinst Corss contaminationNIH terminatescontract of Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler

39、shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Nature calls for aGlobal database ofCell lines STR profiesRoland Nardone starts the Second crusade agsinst Corss contaminationNIH terminatescontract of

40、Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 NIH issues guidelinesTo avoid the use of misidentifi

41、ed cell lines1981-2005 crossContamination spreeds Beyongd HeLa cellsNelson-Ross confirms&estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNature calls for aGlobal database ofCell lines STR profiesRoland Nardone starts the Second crusade agsin

42、st Corss contaminationNIH terminatescontract of Nelson-RossJohn Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 vMor

43、phology check by microscopevGrowth curve analysis vDetection of mycoplasmavSpecies verification by IsoenzymologyvIdentity verification with STR analysisvSpecific properties conformationv Cell cultures received from sources other than recognized cell banks should generally be tested for identity,cont

44、aminations,and suitability of the cells for the assays planed.vThe mycoplasma and cross-contaminations can also occur during cultivation and therefore the cell culture quality tests should be performed at regular intervals.vSimultaneous handling of different cultures under the laminar flow hood by o

45、ne or more persons vthe use of a single flask of medium for different cell lines.vThe prophylactic use of antibiotic v Avoid the negligent and erroneous labelling of cell culture vessels and cryo-ampoules.vUKCCCR Guidelines for the Use of Cell Lines in Cancer Research,British J of Cancer(2000)82(9);

46、14951509.vCell Line Verification Test Recommendations,ATCC Technical Bulletin No.8(2008).vCell Line Authentication Methods vCabrera CM et al.,Identity Test:Determination of Cell Line Cross-Contamination,Cytotechnology(2006)51:4550.vMasters JR et al.,Short Tandem Repeat Profiling Provides an International Reference Standard for Cell Lines,PNAS(2001)98(14):801217.vhttp:/