研究用肿瘤细胞系的质量控制和要求培训课件

1、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.10/7/20221研究用肿瘤细胞系的

2、质量控制和要求Some Facts about the use of cu现代生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 10X10/7/20222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1969200

3、4Common problemsmicrobiological contamination with particular respect to mycoplasma infections (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 o

4、f cell lines leading to a genetic drift of cell cultures and to the selection of altered subclones. 10/7/20223研究用肿瘤细胞系的质量控制和要求Common problemsmicrobiologicalWhere from? faulty cell culture techniques: shared reagents, repeated use of the same pipette during re-feeding operations and manipulation of m

5、ultiple cultures at the same time without adequate isolation of one cell type from another. the introduction of false, contaminated or over-passaged cell lines from other sources into the cell culture laboratory (35% )Mislabeling, lack of awarenessIntentional co-cultivation during propagation of hum

6、an stem or primary cells using a feeder layer derived from another species10/7/20224研究用肿瘤细胞系的质量控制和要求Where from? faulty cell cultur细胞交叉污染历史回顾1968,Apparent HeLa cell contamination of human heteroploid cell lines.Nature.217:750-7511974，HeLa cess contaminating cultures around the world,Sciences 184:1059

7、1981, Contaminated cell cultures. Nature 289:2271981,cross-contamination of cells in culture. Sciences. 212(4493):226-5210/7/20225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,Apparent HeLa c细胞交叉污染历史回顾1999, False human hematopoietic cell lins: cross-cantaminations and misint erpretations. Leukemia.13(10):1601-7200

8、3, 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 database of cell line STR profiles10/7/

9、20226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, False human he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

10、隆）HeLa人宫颈癌ECACC10/7/20227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保藏机构公布的错误细胞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

11、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ATCC10/7/20228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2CHANG Liver人胚胎肝上皮HHAG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

12、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310/7/20229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HeLJOSK-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JC

13、RBL132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410/7/202210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DSMMKN28MKN74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

14、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510/7/202211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CSBC-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S

15、CLCSCLC-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610/7/202212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-7DSMZ 德国细胞库550 leukemia and lymphoma cell lines submitted59/395(15%) by ori

16、ginators23/155(15%) by secondary sources10/7/202213研究用肿瘤细胞系的质量控制和要求DSMZ 德国细胞库550 leukemia and lym交叉污染细胞使用的后果Erroneous results: Wasted research moneyPotential delay in discovery10/7/202214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Erroneous results: 错误细胞使用2000-2004：19/Int 407; 45/WISH; 59/Chang liver; 470/Hep-2; 556

17、/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 used for anticancer drug evaluation. Among them ADR-RES is acutally OVCAR-8. It had been used in about 300 papers.10/7/202215研究用

18、肿瘤细胞系的质量控制和要求错误细胞使用2000-2004：19/Int 407; 45HeLa 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 (2005) WISH (described as “non-transformed amniotic epithelial cells”) in

19、 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, 28106 (2000) HEp-2 (described as “laryngeal cancer”) in Investig. New Dru

20、gs 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 (2005)HeLa, Int-407 and HEp-2 cells were used as three distinct cell lines

21、in the same study in CancerRes. 69, 632 (2009)10/7/202216研究用肿瘤细胞系的质量控制和要求HeLa cervical cancer cells IntEcv-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, can not afford

22、, have to compromise10/7/202217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，ATCCEcv-304/T24 10/7/202218研究用肿瘤细胞系的质量控制和要求Ecv-304/T24 10/3/202218研究用肿瘤细对细胞质量的要求 2007.10,Goslar, Germany，11ICCCScientist from ATCC proposed:(1)funding agencies,gov. or private require cell line authentication as a condition for

23、 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. Authentic, Well characterizedThe problem of Sub-culturing10/7/202219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, GermaNI

24、H NOTICE2007.12,Require cell anthenticationFor grantFor paper10/7/202220研究用肿瘤细胞系的质量控制和要求NIH NOTICE2007.12,10/3/202220研杂志及细胞使用者的要求Examples 1Thp-1, 2009-1-5cell line authentication, characterization and freedom from contamination This is an important issue, to which authors should pay particular atten

25、tion. 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 of contamination (e.g. mycoplasma), and that they have been used from young stock. The

26、se 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 section, and authors omitting to include evidence of good practice will find that their arti

27、cles may be delayed until such guarantees are given. 10/7/202221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Examples 1Thp-1, 2Example 2, Reporting on Cell Line Use in AACR JournalsNEW! EFFECTIVE JULY 1, 2009AACR strongly encourages the authentication of cell lines used in the research reported in its journals. If ce

28、ll 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 cells were tested 4. How and w

29、hen the cells were last tested 10/7/202222研究用肿瘤细胞系的质量控制和要求Example 2, Reporting on Cell LAACR JournalsCancer Research Clinical Cancer Research Cancer Epidemiology, Biomarkers & Prevention， CEBP Cancer Prevention Research Molecular Cancer Research Molecular Cancer Therapeutics Cancer Reviews Online Pr

30、evention Portal 10/7/202223研究用肿瘤细胞系的质量控制和要求AACR JournalsCancer Research 1Example 2shelf 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 r

31、equired. In these cases, please include the method of characterization used by the 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

32、required. In these cases, please include the method of characterization used by the cell bank.10/7/202224研究用肿瘤细胞系的质量控制和要求Example 2shelf lifeIf cells wKey milestones in the effort to address cell line misidentificationHela cell line establishedfrom Henrietta LacksATCC, American Type Culture Collectio

33、n. NIH National Institutes of Health.STR, Short Tandem RepeatInterspecies cross-ContaminationshownATCC starts Curating cell linesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesNelson-Ross confirms & estends Stan Gartlers findingsJohn Maddox describesIndividual like Nelson-

34、RossAs self-appointed vigilantes1981-2005 crossContamination spreeds Beyongd HeLa cellsNIH 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 li

35、nes STR profies1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Hela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNels

36、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 linesNelson-Ross confirms & estends Stan Gartlers

37、 findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell lines1981-2005 crossContamination spreeds Beyongd HeLa cellsNelson-Ross confirms & estends Stan Gartlers findingsHela cell line establishedfrom Henrietta LacksATCC starts Curating cell linesNIH issues guidelinesTo avoid

38、 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 lines1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 Interspecies cross-Contaminat

39、ionshown1952 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 Stan Gartler shows intraspeciesCross-contamination betweenHuman cell linesIntersp

40、ecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantesStan Gartler shows intraspeciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 198

41、1 2005 2007 2009 John 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 terminatescontract of Nelson-RossJohn Madd

42、ox 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 terminatescontract of Nelson-RossJohn Maddox describesIndividual lik

43、e Nelson-RossAs self-appointed vigilantesStan Gartler shows 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 Nels

44、on-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 Roland Nardone starts the Second crusade agsinst Corss c

45、ontaminationNIH 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 Nature calls f

46、or 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 linesI

47、nterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 NIH issues guidelinesTo avoid the use of misidentified cell lines1981-2005 crossContamination spreeds Beyongd HeLa cellsNelson-Ross confirms & estends Stan Gartlers findingsHela cell line establishedfrom Henrietta

48、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 intras

49、peciesCross-contamination betweenHuman cell linesInterspecies cross-Contaminationshown1952 1958 1962 1966 1974 1980 1981 2005 2007 2009 10/7/202225研究用肿瘤细胞系的质量控制和要求Key milestones in the effort tRequirements for cell authentication Morphology check by microscopeGrowth curve analysis Detection of mycoplasmaSpecies verification by IsoenzymologyIdentity verification with STR analysisSpeci