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1、研究用肿瘤细胞系的质量 控制和要求,中国医学科学院基础医学研究所 刘玉琴,Some Facts about the use of cultured cells- Nobel prize,2001，Cell cycle 2002, apoptosis 2006, RNA Interference 2007, homologous recombination and gene targeting 2008,HPVcervical ca, HIV-aids 2009, telomere &telomerase,Nobel prize winner, use in vitro cultured cel
2、ls for their researches.,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 steadily Papers use cells increase 22.5Xbiology, pharmacology and medicine- tumor research 不可少的研究材料、对象、工具 Use of contaminated or misidentified cells increase 10X,Common problems,microbiological contamination with particular
3、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 of cell lines leading to a genetic drift of cell cultures and
4、 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 multiple cultures at the same time without adequate isolation of one cell type from another.the introduction of false
5、, contaminated or over-passaged cell lines from other sources into the cell culture laboratory (35% ) Mislabeling, lack of awareness Intentional 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 conta
6、mination of human heteroploid cell lines.Nature.217:750-751 1974，HeLa cess contaminating cultures around the world,Sciences 184:1059 1981, Contaminated cell cultures. Nature 289:227 1981,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 ce
7、ll lins: cross-cantaminations and misint erpretations. Leukemia.13(10):1601-7 2003, False leukmia lymphoma cell lines :an update on over 500 cell lines. Leukemia 17, 416-426 2004,cell line cross-comtamination:how aware arein vitro cell dev biol anim.40(7):211-5 2007,Cases of mistaken identity.Scienc
8、es 315:928-931 2009, Nature calls for global database of cell line STR profiles,保藏机构公布的错误细胞1,保藏机构公布的错误细胞2,保藏机构公布的错误细胞3,保藏机构公布的错误细胞4,保藏机构公布的错误细胞5,保藏机构公布的错误细胞6,DSMZ 德国细胞库,550 leukemia and lymphoma cell lines submitted 59/395(15%) by originators 23 /155(15%) by secondary sources,交叉污染细胞使用的后果,Erroneous r
9、esults: Wasted research money Potential 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
10、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 (
11、2005)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, 281
12、06 (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 (2005
13、) 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 model Expens
14、ieve, can not afford, have to compromise,Ecv-304/T24,对细胞质量的要求,2007.10,Goslar, Germany，11ICCC Scientist 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
15、. (3) conference, workshop and/or training.(4)lab heads be encouraged to ensure QC measures.Authentic, Well characterized The problem of Sub-culturing,NIH NOTICE,2007.12, Require cell anthentication For grant For paper,杂志及细胞使用者的要求 Examples 1,Thp-1, 2009-1-5 cell line authentication, characterization
16、 and freedom from 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 ens
17、ure 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 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.,
