研究用肿瘤细胞系的质量控制和要求学习培训课件

1、研究用肿瘤细胞系的质量控制和要求Some Facts about the use of cultured cells- Nobel prize 2001，Cell cycle2002, apoptosis2006, RNA Interference2007, homologous recombination and gene targeting2008,HPVcervical ca, HIV-aids2009, telomere &telomeraseNobel prize winner, use in vitro cultured cells for their researches.现代生

2、命科学研究中培养细胞的使用越来越广泛19692004：cell use increasing steadilyPapers use cells increase 22.5X biology, pharmacology and medicine- tumor research不可少的研究材料、对象、工具Use of contaminated or misidentified cells increase 10XCommon problemsmicrobiological contamination with particular respect to mycoplasma infections

3、(incidence of ca. 25%)cross-contamination by other cell lines and misidentification or misclassification (incidence of more than 15%)acquisition 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 subclo

4、nes. Where from? faulty cell culture techniques: 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. the introduction of false, contaminated or over-passaged ce

5、ll lines from other sources into the cell culture laboratory (35% )Mislabeling, lack of awarenessIntentional co-cultivation during propagation of human stem or primary cells using a feeder layer derived from another species细胞交叉污染历史回顾1968,Apparent HeLa cell contamination of human heteroploid cell lin

6、es.Nature.217:750-7511974，HeLa cess contaminating cultures around the world,Sciences 184:10591981, Contaminated cell cultures. Nature 289:2271981,cross-contamination of cells in culture. Sciences. 212(4493):226-52细胞交叉污染历史回顾1999, False human hematopoietic cell lins: cross-cantaminations and misint er

7、pretations. Leukemia.13(10):1601-72003, False leukmia lymphoma cell lines :an update on over 500 cell lines. Leukemia 17, 416-4262004,cell line cross-comtamination:how aware arein vitro cell dev biol anim.40(7):211-52007,Cases of mistaken identity.Sciences 315:928-9312009, Nature calls for global da

8、tabase of cell line STR profiles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保藏机构

9、公布的错误细胞2CHANG 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ATCCGHE人星状细胞瘤T-24

10、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3JOSK-

11、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4MKN28MKN74人JCRBMOLT-15人T细胞白血病CTV-1人单核细胞白

12、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DSMZ保藏机构公布的错误细胞5SBC-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SC

13、LCSCLC-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6DSMZ 德国细胞库550 leukemia and lymphoma cell lines submitted59/395(15%) by originators23/155(15%) by secondary sources交叉污染细胞使用的后果Erroneo

14、us results: Wasted research moneyPotential delay in discovery错误细胞使用2000-2004：19/Int 407; 45/WISH; 59/Chang liver; 470/Hep-2; 556/KB; 2004(Buehring ), pubmed search 220publications involved, 32% use HeLa cells,33% tested for authenticity, 35% get cells from other labs.NCI panel of 60 cell lines were

15、used for anticancer drug evaluation. Among them ADR-RES is acutally OVCAR-8. It had been used in about 300 papers.HeLa cervical cancer cells 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. 280, 13538 (2

16、005) 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) Chang liver (described as “normal liver cells”) in Oncogene 28, 3526 (2009), Proteomics 14, 2885(2008) and J. Biol. Chem. 279, 28

17、106 (2000) HEp-2 (described as “laryngeal cancer”) in Investig. New Drugs 26, 111118 (2008),Carcinogenesis 29, 1519 (2008) and J. Biol. Chem. 283, 36272 (2008)KB (described as “oral cancer”) in Biochem. Pharmacol. 73,19011909 (2007), Clin. Cancer Res. 14,8161(2008) and J. Biol. Chem. 280, 23829 (200

18、5)HeLa, Int-407 and HEp-2 cells were used as three distinct cell lines in the same study in CancerRes. 69, 632 (2009)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19992000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?Donot know? No other suitable cellular modelExpensieve,

19、can not afford, have to compromiseEcv-304/T24 对细胞质量的要求 2007.10,Goslar, Germany，11ICCCScientist 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) co

20、nference, workshop and/or training.(4)lab heads be encouraged to ensure QC measures. Authentic, Well characterizedThe problem of Sub-culturingNIH NOTICE2007.12,Require cell anthenticationFor grantFor paper杂志及细胞使用者的要求Examples 1Thp-1, 2009-1-5cell line authentication, characterization and freedom from

21、 contamination This 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 majority do involve them) are certified as being the designated type, that they have been checked to ensure they are free

22、 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 use of cell lines ( http:/ukcccr.icnet.uk). Attention should be paid to this in the Material and Methods secti

23、on, and authors omitting to include evidence of good practice will find that their articles may be delayed until such guarantees are given. Example 2, Reporting on Cell Line Use in AACR JournalsNEW! EFFECTIVE JULY 1, 2009AACR strongly encourages the authentication of cell lines used in the research

24、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 cell lines have been tested and authenticated 3. The method by which the

25、 cells were tested 4. How and when the cells were last tested AACR JournalsCancer Research Clinical Cancer Research Cancer Epidemiology, Biomarkers & Prevention， CEBP Cancer Prevention Research Molecular Cancer Research Molecular Cancer Therapeutics Cancer Reviews Online Prevention Portal Example 2s

26、helf lifeIf cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6months after receipt or resuscitation, re-authorization is not required. In these cases, please include the method of characterization used by the

27、cell bank.If cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the users laboratory for fewer than 6months after receipt or resuscitation, re-authorization is not required. In these cases, please include the method of characterization used by the

28、 cell bank.Key milestones in the effort to address cell line misidentificationHela cell line establishedfrom Henrietta LacksATCC, American Type Culture Collection. NIH National Institutes of Health.STR, Short Tandem RepeatInterspecies cross-ContaminationshownATCC starts Curating cell linesStan Gartl

29、er 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 issues guid

30、elinesTo 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 Curating cell li

31、nesHela 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 linesNelson-Ross c

32、onfirms & 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 HeLa cel

33、lsNelson-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 fin

34、dingsHela 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 linesIntersp

35、ecies 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-app

36、ointed 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-contamina

37、tion 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 lin

38、esInterspecies 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-Conta

39、minationshown1952 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 betwe

40、enHuman 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 s

41、hows 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 N

42、elson-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 misidentifie

43、d 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 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 1966 1974 1980