研究用腫瘤細(xì)胞系的質(zhì)量控制和要求

1、研究用腫瘤細(xì)胞系的質(zhì)量控制和要求,中國(guó)醫(yī)學(xué)科學(xué)院基礎(chǔ)醫(yī)學(xué)研究所劉玉琴,Some Facts about the use of cultured cells-- Nobel prize,2001，Cell cycle2002, apoptosis2006, RNA Interference2007, homologous recombination and gene targeting2008,HPV—cervical ca,

2、 HIV--aids2009, telomere &telomerase,Nobel prize winner, use in vitro cultured cells for their researches.,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,1969~2004：cell use increasing steadilyPapers use cells increase 2~2.5X biology, phar

3、macology and medicine---- tumor research不可少的研究材料、對(duì)象、工具Use of contaminated or misidentified cells increase 10X,Common problems,microbiological contamination with particular respect to mycoplasma infections (incidence o

4、f 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

5、 genetic drift of cell cultures and to the selection of altered subclones.,Where from?,faulty cell culture techniques: shared reagents, repeated use of the same pipette during re-feeding operations and manipulation of mu

6、ltiple 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% )M

7、islabeling, lack of awarenessIntentional co-cultivation during propagation of human stem or primary cells using a feeder layer derived from another species,細(xì)胞交叉污染歷史回顧,1968,Apparent HeLa cell contamination of human heter

8、oploid cell lines.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(4

9、493):226-52,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):1601-72003, False leukmia –lymphoma cell lines :an update on over 500 cell lines. Leukemia

10、17, 416-4262004,cell line cross-comtamination:how aware are…in 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 profiles,保

11、藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞1,保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞2,保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞3,保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞4,保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞5,保藏機(jī)構(gòu)公布的錯(cuò)誤細(xì)胞6,DSMZ 德國(guó)細(xì)胞庫(kù),550 leukemia and lymphoma cell lines submitted59/395(15%) by originators23/155(15%) by secondary sources,交叉污染細(xì)胞使用的后果,Erroneous re

12、sults: Wasted research moneyPotential delay in discovery,錯(cuò)誤細(xì)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

13、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.,HeLa cervical cancer ce

14、lls,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 epithe

15、lial 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) a

16、nd J. Biol. Chem. 279, 28106 (2000) HEp-2 (described as “l(fā)aryngeal cancer”) in Investig. New Drugs 26, 111–118 (2008),Carcinogenesis 29, 1519 (2008) and J. Biol. Chem. 283, 36272 (2008)KB (described as “oral cancer”) i

17、n Biochem. Pharmacol. 73,1901–1909 (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 in the same study in CancerRes. 69, 632

18、 (2009),Ecv-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1999—2000年，經(jīng)STR檢測(cè)，證明為T(mén)24(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, have t

19、o compromise,Ecv-304/T24,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 the award of grant and contracts funds

20、. (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-culturing,NIH NOTICE,20

21、07.12,Require cell anthenticationFor grantFor paper,雜志及細(xì)胞使用者的要求Examples 1,Thp-1, 2009-1-5cell line authentication, characterization and freedom from contamination This is an important issue, to which authors should

22、 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 e

23、nsure 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 use of cell

24、 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 gu

25、arantees are given.,Example 2, Reporting on Cell Line Use in AACR Journals,NEW! EFFECTIVE JULY 1, 2009  AACR strongly encourages the authentication of cell lines used in the research reported in its journals.

26、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 obtai

27、ned 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

28、 tested,AACR Journals,Cancer Research Clinical Cancer Research Cancer Epidemiology, Biomarkers & Prevention， CEBP Cancer Prevention Research Molecular Cancer Research Molecular Cancer Therapeutics Cancer Review

29、s Online Prevention Portal,Example 2—shelf life,If cells were obtained directly from a cell bank that performs cell line characterizations and passaged in the user's laboratory for fewer than 6 months after rec

30、eipt or resuscitation, re-authorization is not required. 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 chara

31、cterizations and passaged in the user's laboratory for fewer than 6 months after receipt or resuscitation, re-authorization is not required. In these cases, please include the method of characterization used by

32、the cell bank.,Key milestones in the effort to address cell line misidentification,Hela cell line establishedfrom Henrietta Lacks,ATCC, American Type Culture Collection. NIH National Institutes of Health.STR, Short Tan

33、dem Repeat,Interspecies cross-Contaminationshown,ATCC starts Curating cell lines,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Nelson-Ross confirms & estends Stan Gartler’s findings

34、,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,1981-2005 crossContamination spreeds Beyongd HeLa cells,NIH terminatescontract of Nelson-Ross,NIH issues guidelinesTo avoid the use of

35、misidentified cell lines,Roland Nardone starts the Second crusade agsinst Corss contamination,Nature calls for aGlobal database ofCell lines STR profies,,1952 1958 1962 1966 1974 1980 1981

36、 2005 2007 2009,,,,,,,,,,,,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,,,Hela cell line e

37、stablishedfrom Henrietta Lacks,ATCC starts Curating cell lines,Nelson-Ross confirms & estends Stan Gartler’s findings,,,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,,Nelson-Ross

38、 confirms & estends Stan Gartler’s findings,,,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,,,Nelson-Ross confirms & estends Stan Gartler’s findings,,,Hela cell line establish

39、edfrom Henrietta Lacks,ATCC starts Curating cell lines,1981-2005 crossContamination spreeds Beyongd HeLa cells,,,Nelson-Ross confirms & estends Stan Gartler’s findings,,,Hela cell line establishedfrom Henrietta

40、 Lacks,ATCC starts Curating cell lines,NIH issues guidelinesTo avoid the use of misidentified cell lines,1981-2005 crossContamination spreeds Beyongd HeLa cells,,,Nelson-Ross confirms & estends Stan Gartler’s f

41、indings,,,Hela cell line establishedfrom Henrietta Lacks,ATCC starts Curating cell lines,,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,Interspecies cross-Contaminationshow

42、n,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 195

43、8 1962 1966 1974 1980 1981 2005 2007 2009,,,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962

44、 1966 1974 1980 1981 2005 2007 2009,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell li

45、nes,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan

46、Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,NIH terminatescon

47、tract of Nelson-Ross,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,

48、1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,,NIH terminatescontract of Nelson-Ross,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gar

49、tler shows intraspeciesCross-contamination betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,Roland Nardone starts

50、 the Second crusade agsinst Corss contamination,,NIH terminatescontract of Nelson-Ross,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gartler shows intraspeciesCross-contaminat

51、ion betweenHuman cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,,Roland Nardone starts the Second crusade agsinst Corss con

52、tamination,,NIH terminatescontract of Nelson-Ross,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gartler shows intraspeciesCross-contamination betweenHuman cell lines,Interspeci

53、es cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,Nature calls for aGlobal database ofCell lines STR profies,,Roland Nardone starts the Second crus

54、ade agsinst Corss contamination,,NIH terminatescontract of Nelson-Ross,,John Maddox describesIndividual like Nelson-RossAs self-appointed vigilantes,,Stan Gartler shows intraspeciesCross-contamination betweenHuman

55、 cell lines,Interspecies cross-Contaminationshown,1952 1958 1962 1966 1974 1980 1981 2005 2007 2009,,,Requirements for cell authentication,Morphology check by microscopeGrowth cu

56、rve analysis Detection of mycoplasmaSpecies verification by IsoenzymologyIdentity verification with STR analysisSpecific properties conformation,Detection of cross-comtamination,STR PROFILING METHODOLOGY,ADVANTAGES,,

57、How to avoid cross-contaminations,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. The myc

58、oplasma and cross-contaminations can also occur during cultivation and therefore the cell culture quality tests should be performed at regular intervals. Simultaneous handling of different cultures under the laminar f

59、low hood by one or more persons the use of a single flask of medium for different cell lines. The prophylactic use of antibiotic Avoid the negligent and erroneous labelling of cell culture vessels and cryo-ampoules.

60、,Resources for Authors,UKCCCR Guidelines for the Use of Cell Lines in Cancer Research, British J of Cancer (2000) 82(9); 1495–1509. Cell Line Verification Test Recommendations, ATCC Technical Bulletin No. 8 (2008).

61、 Cell Line Authentication Methods Cabrera CM et al., Identity Test: Determination of Cell Line Cross-Contamination, Cytotechnology (2006) 51:45–50. Masters JR et al., Short Tandem Repeat Profiling Provides an Internat