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.11/5/20221研究用肿瘤细胞系的质量控制和要求v19692004：cell use increasing steadilyvPapers use
2、cells increase 22.5Xv biology,pharmacology and medicine-tumor researchv不可少的研究材料、对象、工具vUse of contaminated or misidentified cells increase 10X11/5/20222研究用肿瘤细胞系的质量控制和要求vmicrobiological contamination with particular respect to mycoplasma infections(incidence of ca.25%)vcross-contamination by other cel
3、l lines and misidentification or misclassification(incidence 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.11/5/20223研究用肿瘤细胞系的质量控制和要求v faulty cell culture te
4、chniques:shared reagents,repeated use of the same pipette during re-feeding operations 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
5、 culture laboratory(35%)vMislabeling,lack of awarenessvIntentional co-cultivation during propagation of human stem or primary cells using a feeder layer derived from another species11/5/20224研究用肿瘤细胞系的质量控制和要求v1968,Apparent HeLa cell contamination of human heteroploid cell lines.Nature.217:750-751v197
6、4，HeLa cess contaminating cultures around the world,Sciences 184:1059v1981,Contaminated cell cultures.Nature 289:227v1981,cross-contamination of cells in culture.Sciences.212(4493):226-5211/5/20225研究用肿瘤细胞系的质量控制和要求v1999,False human hematopoietic cell lins:cross-cantaminations and misint erpretations.
7、Leukemia.13(10):1601-7v2003,False leukmia lymphoma cell lines:an update on over 500 cell lines.Leukemia 17,416-426v2004,cell line 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 cel
8、l line STR profiles11/5/20226研究用肿瘤细胞系的质量控制和要求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ECACC11/
9、5/20227研究用肿瘤细胞系的质量控制和要求CHANG 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ATC
10、CGHE人星状细胞瘤T-24 人膀胱癌DSMZGirardi Heart猪成年心脏HeLa人宫颈癌ATCC11/5/20228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DSMZIntestine 407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
11、单核细胞白血病U-937人组织细胞淋巴瘤DSMZ11/5/20229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D
12、SMZ11/5/202210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DSMZ11/5/202211研究用肿瘤细胞系的质量控制和要求SBC-2人膀胱癌
13、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ATCC11/5/202212研究用肿瘤细胞系的质量控制和要求v550 leukemia and lymphoma cell
14、lines submittedv59/395(15%)by originatorsv23/155(15%)by secondary sources11/5/202213研究用肿瘤细胞系的质量控制和要求vErroneous results:vWasted research moneyvPotential delay in discovery11/5/202214研究用肿瘤细胞系的质量控制和要求v2000-2004：19/Int 407;45/WISH;59/Chang liver;470/Hep-2;556/KB;v2004(Buehring),pubmed search 220publicat
15、ions involved,32%use HeLa cells,33%tested for authenticity,35%get cells 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.11/5/202215研究用肿瘤细胞系的质量控制和要求v Int-407(described as“non-transformed i
16、ntestinal epithelial cells”)in Br.J.Cancer 101,1596(2009),EMBO J.22,5003(2003)and J.Biol.Chem.280,13538(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
17、 cells”)in Oncogene 28,3526(2009),Proteomics 14,2885(2008)and J.Biol.Chem.279,28106(2000)v HEp-2(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.C
18、ancer Res.14,8161(2008)and J.Biol.Chem.280,23829(2005)vHeLa,Int-407 and HEp-2 cells were used as three distinct cell lines in the same study in CancerRes.69,632(2009)11/5/202216研究用肿瘤细胞系的质量控制和要求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
19、。Why?vDonot know?vNo other suitable cellular modelvExpensieve,can not afford,have to compromise11/5/202217研究用肿瘤细胞系的质量控制和要求11/5/202218研究用肿瘤细胞系的质量控制和要求v2007.10,Goslar,Germany，11ICCCvScientist from ATCC proposed:(1)funding agencies,gov.or private require cell line authentication as a condition for the
20、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 Authentic,Well characterizedvThe problem of Sub-culturing11/5/202219研究用肿瘤细胞系的质量控制和要求v2007.12,vRequire cell anthenticationvFo
21、r grantvFor paper11/5/202220研究用肿瘤细胞系的质量控制和要求vThp-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 responsibility to ensure that cell lines used in any study(and the major
22、ity 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 which good practice is based,as set out in the UKCCC Guidelines on the u
23、se 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 guarantees are given.11/5/202221研究用肿瘤细胞系的质量控制和要求vNEW!EFFECTIVE JULY 1,2009 vA
24、ACR 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 when the cells were obtained 2.Whether the
25、 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 11/5/202222研究用肿瘤细胞系的质量控制和要求vCancer Research vClinical Cancer Research vCancer Epidemiology,Biomarkers&Prevention，CEBP vCancer Prevention Research vMolecular Cancer Res
26、earch vMolecular Cancer Therapeutics vCancer Reviews Online vPrevention Portal 11/5/202223研究用肿瘤细胞系的质量控制和要求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-authori
27、zation is not 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-authori
28、zation is not required.In these cases,please include the method of characterization used by the cell bank.11/5/202224研究用肿瘤细胞系的质量控制和要求Hela cell line establishedfrom Henrietta LacksATCC,American Type Culture Collection.NIH National Institutes of Health.STR,Short Tandem RepeatInterspecies cross-Contami
29、nationshownATCC starts Curating cell linesStan 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 cellsNI
30、H terminatescontract of Nelson-RossNIH 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 establish
31、edfrom Henrietta LacksATCC starts Curating 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 L
32、acksATCC starts Curating cell linesNelson-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
33、 crossContamination spreeds Beyongd HeLa 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
34、-Ross confirms&estends Stan Gartlers findingsHela 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-cont
35、amination betweenHuman cell linesInterspecies 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 describ
36、esIndividual like Nelson-RossAs self-appointed 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 G
37、artler shows intraspeciesCross-contamination 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 intraspeciesC
38、ross-contamination 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 between
39、Human 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 sho
40、ws intraspeciesCross-contamination betweenHuman 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
41、 self-appointed vigilantesStan Gartler 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
42、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 NIH issues gu
43、idelinesTo avoid the use of misidentified 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
44、Nardone starts the Second crusade agsinst 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
45、1966 1974 1980 1981 2005 2007 2009 11/5/202225研究用肿瘤细胞系的质量控制和要求vMorphology check by microscopevGrowth curve analysis vDetection of mycoplasmavSpecies verification by IsoenzymologyvIdentity verification with STR analysisvSpecific properties conformation11/5/202226研究用肿瘤细胞系的质量控制和要求11/5/202227研究用肿瘤细胞系的质量
46、控制和要求11/5/202228研究用肿瘤细胞系的质量控制和要求11/5/202229研究用肿瘤细胞系的质量控制和要求v Cell cultures received from sources other than recognized cell banks should generally be tested for identity,contaminations,and suitability of the cells for the assays planed.vThe mycoplasma and cross-contaminations can also occur during c
47、ultivation and therefore the cell culture quality tests should be performed at regular intervals.vSimultaneous handling of different cultures under the laminar flow hood by one or more persons vthe use of a single flask of medium for different cell lines.vThe prophylactic use of antibiotic v Avoid t
48、he negligent and erroneous labelling of cell culture vessels and cryo-ampoules.11/5/202230研究用肿瘤细胞系的质量控制和要求vUKCCCR Guidelines for the Use of Cell Lines in Cancer Research,British J of Cancer(2000)82(9);14951509.vCell Line Verification Test Recommendations,ATCC Technical Bulletin No.8(2008).vCell Line
49、 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:/11/5/202231研究用肿瘤细胞系的质量控制和要求 11/5/202232研究用肿瘤细胞系的质量控制和要求
