研究用腫瘤細(xì)胞系的質(zhì)量控制和要求培訓(xùn)課件

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研究用腫瘤細(xì)胞系的

2、質(zhì)量控制和要求Some Facts about the use of cu現(xiàn)代生命科學(xué)研究中培養(yǎng)細(xì)胞的使用越來(lái)越廣泛19692004：cell use increasing steadilyPapers use cells increase 22.5X biology, pharmacology and medicine- tumor research不可少的研究材料、對(duì)象、工具Use of contaminated or misidentified cells increase 10X10/7/20222研究用腫瘤細(xì)胞系的質(zhì)量控制和要求現(xiàn)代生命科學(xué)研究中培養(yǎng)細(xì)胞的使用越來(lái)越廣泛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研究用腫瘤細(xì)胞系的質(zhì)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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求Where from? faulty cell cultur細(xì)胞交叉污染歷史回顧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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求細(xì)胞交叉污染歷史回顧1968,Apparent HeLa c細(xì)胞交叉污染歷史回顧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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求細(xì)胞交叉污染歷史回顧1999, False human he保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞1細(xì)胞系種屬細(xì)胞類型污染源種屬細(xì)胞類型數(shù)據(jù)來(lái)源207人前B白血病REH人前B白血病DSMZ2474/90人胃癌HT-29人結(jié)直腸癌DSMZ2957/90人胃癌HT-29人結(jié)直腸癌DSMZ3051/80人胃癌HT-29人結(jié)直腸癌DSMZADLC-5M人肺癌HELA/-S3人宮頸癌DSMZAV3人羊膜HeLa人宮頸癌ATCCBCC-1/KMC人基底細(xì)胞癌HELA/-S3人宮頸癌DSMZBM-1640人前列腺癌DU-145人前列腺癌DSMZC16人胚胎肺成纖維細(xì)胞（MRC-5克

10、?。〩eLa人宮頸癌ECACC10/7/20227研究用腫瘤細(xì)胞系的質(zhì)量控制和要求保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞1細(xì)胞系種屬細(xì)胞類型污染源種屬細(xì)胞類型保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞2CHANG Liver人胚胎肝上皮HeLa人宮頸癌ATCC;JCRBCOLO-818人黑色素瘤COLO800人黑色素瘤DSMZDAMI人巨核細(xì)胞HEL人紅系白血病DSMZECV304人內(nèi)皮T24人膀胱癌ATCCECV-304人正常內(nèi)皮T24人膀胱癌DSMZEJ人膀胱癌T24人膀胱癌ATCC;JCRBEPLC3-2M1人肺癌HELA/-S3人宮頸癌DSMZ;JCRBEPLC-65人肺癌HELA/-S3人宮頸癌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人宮頸癌ATCCGHE人星狀細(xì)胞瘤T-24 人膀胱癌DSMZGirardi Heart豬成年心臟HeLa人宮頸癌ATCC10/7/20228研究用腫瘤細(xì)胞系的質(zhì)量控制和要求保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)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人宮頸癌ATCCHMV-1人黑色素瘤HeLa-S3人宮頸癌DSMZHuL-1HeLa人宮頸癌JCRBIMC-2人顎骨癌HeLa-S3人宮頸癌DSMZIntestine 407人小腸上皮HeLa人宮頸癌

12、ATCCJ-111HeLa人宮頸癌JCRBJOSK-I人單核細(xì)胞白血病U-937人組織細(xì)胞淋巴瘤DSMZJOSK-K人單核細(xì)胞白血病U-937人組織細(xì)胞淋巴瘤DSMZJOSK-M人單核細(xì)胞白血病U-937人組織細(xì)胞淋巴瘤DSMZ保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞310/7/20229研究用腫瘤細(xì)胞系的質(zhì)量控制和要求HAG人T-24人膀胱癌DSMZHEp-2人成年喉上皮HeLJOSK-S人單核細(xì)胞白血病U-937人組織細(xì)胞淋巴瘤DSMZJTC-17 HeLa人宮頸癌Yamakage(JCRB)KB人HeLa人宮頸癌ATCC;JCRBKO51K562人髓系白血病DSMZ;JCRBKOSC-3Ca9-22人JC

13、RBL132人胚胎肺上皮HeLa人宮頸癌ATCCLR10.6人前B白血病NALM-6人前B 白血病DSMZMaTu人乳腺癌HELA/-S3人宮頸癌DSMZMC-4000人乳腺癌HELA/-S3人宮頸癌DSMZMKB-1人T細(xì)胞白血病CCRF-CEM人T細(xì)胞白血病DSMZ保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞410/7/202210研究用腫瘤細(xì)胞系的質(zhì)量控制和要求JOSK-S人單核細(xì)胞白血病U-937人組織細(xì)胞淋巴瘤DSMMKN28MKN74人JCRBMOLT-15人T細(xì)胞白血病CTV-1人單核細(xì)胞白血病DSMZMT-1人乳腺癌HELA/-S3人宮頸癌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人成纖維WI38人成纖維JCRBRAMAK-1人肌肉滑膜T-24人膀胱癌DSMZ保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞510/7/202211研究用腫瘤細(xì)胞系的質(zhì)量控制和要求MKN28MKN74人JCRBMOLT-15人T細(xì)胞白血病CSBC-2人膀胱癌HELA/-S3人宮頸癌DSMZSBC-7人膀胱癌HELA/-S3人宮頸癌DSMZSCLC-16H人SCLCSCLC-21/22H人SCLCDSMZSCLC-24H人S

15、CLCSCLC-21/22H人SCLCDSMZSNB-19U251MG人膠質(zhì)瘤ATCCSPI-801人T細(xì)胞白血病K562人髓系白血病DSMZSPI-802人T細(xì)胞白血病K562人髓系白血病DSMZTK-1人?U251MG人膠質(zhì)瘤JCRBTMH-1IHH-4人JCRBWISH人新生羊膜上皮HeLa人宮頸癌ATCC保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞610/7/202212研究用腫瘤細(xì)胞系的質(zhì)量控制和要求SBC-2人膀胱癌HELA/-S3人宮頸癌DSMZSBC-7DSMZ 德國(guó)細(xì)胞庫(kù)550 leukemia and lymphoma cell lines submitted59/395(15%) by ori

16、ginators23/155(15%) by secondary sources10/7/202213研究用腫瘤細(xì)胞系的質(zhì)量控制和要求DSMZ 德國(guó)細(xì)胞庫(kù)550 leukemia and lym交叉污染細(xì)胞使用的后果Erroneous results: Wasted research moneyPotential delay in discovery10/7/202214研究用腫瘤細(xì)胞系的質(zhì)量控制和要求交叉污染細(xì)胞使用的后果Erroneous results: 錯(cuò)誤細(xì)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、腫瘤細(xì)胞系的質(zhì)量控制和要求錯(cuò)誤細(xì)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 “l(fā)aryngeal 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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求HeLa cervical cancer cells IntEcv-304,“人臍靜脈內(nèi)皮細(xì)胞”日本實(shí)驗(yàn)室建立，ATCC、DSMZ等機(jī)構(gòu)有保藏，在19992000年，經(jīng)STR檢測(cè)，證明為T24(膀胱癌)，各自網(wǎng)站有公布。在實(shí)驗(yàn)室間廣泛傳播，2006y,全世界600余論文，利用該細(xì)胞進(jìn)行研究，2/3在中國(guó)。Why?Donot know? No other suitable cellular modelExpensieve, can not afford

22、, have to compromise10/7/202217研究用腫瘤細(xì)胞系的質(zhì)量控制和要求Ecv-304,“人臍靜脈內(nèi)皮細(xì)胞”日本實(shí)驗(yàn)室建立，ATCCEcv-304/T24 10/7/202218研究用腫瘤細(xì)胞系的質(zhì)量控制和要求Ecv-304/T24 10/3/202218研究用腫瘤細(xì)對(duì)細(xì)胞質(zhì)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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求對(duì)細(xì)胞質(zhì)量的要求 2007.10,Goslar, GermaNI

24、H NOTICE2007.12,Require cell anthenticationFor grantFor paper10/7/202220研究用腫瘤細(xì)胞系的質(zhì)量控制和要求NIH NOTICE2007.12,10/3/202220研雜志及細(xì)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研究用腫瘤細(xì)胞系的質(zhì)量控制和要求雜志及細(xì)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研究用腫瘤細(xì)胞系的質(zhì)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研究用腫瘤細(xì)胞系的質(zhì)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研究用腫瘤細(xì)胞系的質(zhì)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研究用腫瘤細(xì)胞系的質(zhì)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