Cell Line Authentication - PowerPoint Presentation

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Cell Line Authentication

ICLAC2021

Challenges of using cell lines in biomedical research

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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.

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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

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A History of Addressing Cell Line Misidentification

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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

Case 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

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Kolon

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!

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Minimal 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

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Geraghty, R. J., et al. (2014). "Guidelines for the use of cell lines in biomedical research." Br J Cancer

111

(6): 1021-1046.

Goals

: (1) to uniquely distinguish cell lines derived from individual donors (2) to monitor stability of general cell characteristics

Cell Line Authentication = Data Reproducibility

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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

Butler,

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

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Butler,

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

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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

a

cgta

cgtacgtacg

tac

Simple Solutions to Ensure Data Integrity, Reproducibility: STR

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Now 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

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Good 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

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ICLAC

(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

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Acknowledging Leaders Fighting for Authentication

Stan

Gartler

Walter Nelson-Rees

1929-2009

1923-

Roland Nardone

1928-2018

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ICLAC:

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

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STR 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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