ICLAC 2021 Challenges of using cell lines in biomedical research 2 Authentication Misidentification Intraspecies crosscontamination Interspecies crosscontamination 1530 ID: 919654
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Cell Line Authentication
ICLAC2021
Slide2Challenges of using cell lines in biomedical research
2
Authentication Misidentification Intra-species cross-contamination Inter-species cross-contamination 15-30%
Annotation
Cell line names
Associated terms (Pathology
etc
) Contamination Adventitious organisms (Mycoplasma etc) ~20% Best Practices Adherence to best cell culture techniques “Drift”, divergence
-Lewis L.
Coriell, Natl Cancer Inst Monogr. 1962 Apr;7:33-53.
Slide33
530 widely used cell lines are misidentified
Can be wrong tissue, even wrong species15-30% of all cell lines misidentified or contaminated
~
50%
of researchers do not authenticate cell lines
The Depth of the Problem
Slide44
A History of Addressing Cell Line Misidentification
Slide55
Up to 57% of food is mislabeled
Khaksar, R., et al. (2015). "Unmasking seafood mislabeling in U.S. markets: DNA barcoding as a unique technology for food authentication and quality control." Food Control 56: 71-76.
Di Pinto, A., et al. (2015). "Occurrence of mislabeling in meat products using DNA-based assay." Journal of food science and technology 52(4): 2479-2484.
Pardo, M. Á., et al. (2018). "DNA barcoding revealing mislabeling of seafood in European mass caterings." Food Control 92: 7-16.
Why is Authentication Important?
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Think of the consequences…..
Why is Authentication Important?
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Nature 505, 212–217 (09 January 2014)
REPUTATION
Nature. 2012 Mar 28;483(7391):531-3
Amgen validates only 6/53 (11%) “landmark” paper findings
Other studies: 22-49%
TRANSLATION
PLoS
Biol
13(6): e1002165
Prevalence of irreproducible preclinical research exceeds 50%
~$28 billion in funding wasted per year
COST $$$
Retraction of work
Loss of reputation and credibility
Clinical trials halted
The Consequences of Not Getting it Right
Slide8Case Study – HEp-2
HEp-2 (described as epidermoid carcinoma of the larynx)
Is in fact HeLa (identified as contaminated in 1966)
Clearly stated on ATCC website (but available- ATCC
®
CCL-23™)
Latest paper on HEp-2:
Systemic failure by authors, reviewers and the journals (>5,000 articles contain HEp-2)
* Gartler, S.M. 1967. NCI Monograph 26: Second Decennial Review Conference on Cell. Tissue and Organ Culture. NCI, Bethesda, MD:167-195. * Science. 2015 Feb 27;347(6225):938-40 (Christopher Korch)
Adv Clin Exp Med
.
2020
;29(5):525–534
8
Slide9Kolon
Life Science’s drug InvossaCell-based therapy for osteoarthritisFound to be 293 cells, not cartilage cellsKorean government revoked approval for mislabeling and false reporting of ingredientsHad to repay $39.1 million to Mitsubishi PharmaFDA suspended patient enrollment for Phase III clinical trial (subsequently lifted)438 hospitals and clinics have administered 3,707 doses of the drug so far (>3,000
patients)Patients need 15 year follow up (cost: $66.9 million)
Case Study –
Invossa
Avoidable with a ~$100 STR test!
9
Slide10Minimal Requirements for cell line authentication and quality control
Source Obtain cell lines from a reputable source If infeasible, perform rigorous quality controls before using
Identity DNA-based test of origin (compared to reference): STR, SNP Test for species (COI)Purity Test for microbial contamination (Mycoplasma, fungi, bacteria, virus) DNA-based test (STR, SNP)
Functionality
Test unique characteristic related to study
Visual inspection (phase microscopy) for morphology, contamination
DNA-based origin tests should be performed frequently
Mycoplasma tests should be performed on a monthly basisVisual inspection should be performed each time cultures are handled
Simple Solutions to Ensure Data Integrity, Reproducibility
10
Geraghty, R. J., et al. (2014). "Guidelines for the use of cell lines in biomedical research." Br J Cancer
111
(6): 1021-1046.
Slide11Goals
: (1) to uniquely distinguish cell lines derived from individual donors (2) to monitor stability of general cell characteristics
Cell Line Authentication = Data Reproducibility
11
Technique
Application
DNA Fingerprinting –
STR
, SNP, CGH, Sequencing
IndividualityCytogenetics- Karyotyping, FISH, SKY, G-bandingIndividuality; species
Isoenzyme Analysis
Species
Morphology, growth, cell cycle
Phenotypic
Techniques used to authenticate cell lines
Slide12Butler,
Biotechniques. 2007 Oct;43(4):ii-v.
Short, 2-7bp polymorphic sequences of DNA
Often referred to as microsatellites
Repeated multiple (~6-several dozen) times head-to-tail
e.g.
g
t
acgt
acgtacgta
c
g
t
a
c
Simple Solutions to Ensure Data Integrity, Reproducibility: STR
12
Slide13Butler,
Biotechniques. 2007 Oct;43(4):ii-v.
Short, 2-7bp polymorphic sequences of DNA
Often referred to as microsatellites
Repeated multiple (~6-several dozen) times head-to-tail
e.g.
g
t
acgtacgta
cgtacgta
c
Simple Solutions to Ensure Data Integrity, Reproducibility: STR
13
Slide14Short, 2-7bp polymorphic sequences of DNA
Often referred to as microsatellites
Repeated multiple (~6-several dozen) times head-to-tail
e.g.
g
t
a
cgta
cgtacgtacg
tac
Simple Solutions to Ensure Data Integrity, Reproducibility: STR
14
Slide15Now I have an STR Profile, What Do I Do?
Hep-2 STR
Result
https://web.expasy.org/cellosaurus-str-search
Can upload multiple STR profiles to compare
>95% match to HeLa
15
Slide16Good Handling Guidelines
Get cells from reputable sourceFollow good lab practices/SOPsRegularly thaw cells from stocksWork with one cell line at a timeBe detail-oriented (labeling etc.)Regularly test for mycoplasma
Check ICLAC and Cellosaurus!!When to authenticateObtaining cells from another lab
Cells from unreliable source
Old, unverified stocks
After freezing new stocks
After selection (e.g. drug resistance)
Cells propagated for long time
Simple Guidelines to Maintain Authentic Cultures
16
Slide17ICLAC
(International Cell Line Authentication Committee)https://iclac.org/Register of Misidentified Cell Lines:https://iclac.org/databases/cross-contaminations/Cellosaurushttps://web.expasy.org/cellosaurus/Knowledge resource on cell linesLots of great info on cell lines!
ATCC Standards Development Organization (SDO) ASN-0002: Authentication of Human Cell Lines: Standardization of STR Profilinghttps://webstore.ansi.org/Standards/ATCC/ANSIATCCASN00022011Lots of great advice on how to perform cell cultureOECD Guidance Document on Good In Vitro Method Practices (GIVIMP)https://doi.org/10.1787/9789264304796-en
Resources
17
Slide18Acknowledging Leaders Fighting for Authentication
Stan
Gartler
Walter Nelson-Rees
1929-2009
1923-
Roland Nardone
1928-2018
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Slide20ICLAC:
http://iclac.org/Database of misidentified lines: http://iclac.org/databases/cross-contaminations/Guidelines to authentication: http://iclac.org/resources/human-cell-line-authentication/ATCC STR profiling: http://www.atcc.org/Products/Cells_and_Microorganisms/Testing_and_Characterization/STR_Profiling_Analysis.aspx
Papers:Geraghty, R. J., et al. (2014). "Guidelines for the use of cell lines in biomedical research." Br J Cancer 111(6): 1021-1046. http://www.ncbi.nlm.nih.gov/pubmed/25117809Freedam et al (2015) The culture of cell culture practices and authentication--Results from a 2015 Survey. Biotechniques. 2015 Oct 1;59(4):189-90, 192.
http://www.ncbi.nlm.nih.gov/pubmed/26458546
Yu et al. (2015) A resource for cell line authentication, annotation and quality control.
Nature. 2015
Apr
16;520(7547):307-11http://www.ncbi.nlm.nih.gov/pubmed/25877200Liang-Chu et al. (2015) Human biosample authentication using the high-throughput, cost-effective SNPtrace(TM) system. PLoS One. 2015 Feb 25;10(2):e0116218
http://www.ncbi.nlm.nih.gov/pubmed/25714623Capes-Davis et al (2013) Match criteria for human cell line authentication: where do we draw the line? Int J Cancer. 2013 Jun 1;132(11):2510-9 http://www.ncbi.nlm.nih.gov/pubmed/23136038
Useful Links / Resources
20
Slide21STR and SNP can detect human
intra-species contaminationCurrently no commercial options to authenticate non-human cell linesCross-species contamination is not detected by SNP or STR
In Vitro Cell.Dev.Biol.—Animal (2007) 43:344–351
Multiplex PCR-based assay
Mitochondrial cytochrome C oxidase I gene (COI)
Detecting Cross-Species Contamination
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