Misidentification of human cell lines:
Science vs. Policy
Yvonne A. Reid, PhD
Manager, Scientist, Cell Biology Program
CELL Culture 2012, San Diego, CA
Date: 02/03/2012
Outline
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History of misidentified cell lines
Responsibilities of stakeholders
STR as the ‘gold standard’ for human
cell line identification
STR profile testing
Steps to reduce misidentified cell
lines
1952: HeLa - First human cancer cell line was
derived
HeLa cell line (ATCC® CCL-2™)
derived from a glandular cervical
cancer
George Gey, Mary Kubicek
Johns Hopkins University
Hospital, Baltimore, MD
Gey, GO et al. Cancer Res. 12:264, 1952
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HeLa – Henrietta Lacks
31 year-old mother of 4 children,
Roanoke, VA.
1950s: Primitive tissue culture practices
lead to cross-contamination
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No laminar flow hoods
No plastics
No commercial media
• beef embryo extracts
• human cord blood
• chick plasma
1959: Proposal for standardized collection of
animal cell lines
• 1959:
NCI proposes standardized
collection of animal cell lines in an
effort to reduce widespread
contamination and misidentification
among cell lines used in research
Georgetown, DC,
1956
• 1962:
American Type Culture
Collection (ATCC) appointed as
repository for the storage,
authentication and distribution of
animal cell lines. Cell Biology
Collection was established.
Rockville, MD, 1964
Manassas, VA, 1998
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1967 and 1968: Stanley Gartler describes
intraspecies cross-contamination
Isoenzyme Analysis
Type A (fast)
Glucose-6-phosphate dehydrogenase (G6PD) Type B (slow)
Origin
Name
Description
ATCC catalog
no.
Origin
G6PD variant
HeLa
Cervical adenocarcinoma; human
ATCC®CCL-2™
African
Type A (fast)
KB
Oral epidermoid carcinoma, human
ATCC®CCL-17™
Caucasian
Type A (fast)
HEp-2
Larynx epidermoid carcinoma,
human
ATCC®CCL-23™
Caucasian
Type A (fast)
Chang
liver
Liver, human
ATCC®CCL-13™
Caucasian
Type A (fast)
Int-407
Embryonic intestine; human
ATCC®CCL-6™
Caucasian
Type A (fast)
Conclusion: 90% (18/20) human cell lines are ‘HeLa’, later confirmed
by karyotyping and DNA fingerprinting analyses
Gartler SM, NCI Monogr. 26:176, 1967; Gartler, SM, Nature 217:750, 1968
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1981: Walter Nelson-Rees describes
interspecies cross- contamination
Actual
(43/466 (9.2%))
Purported
(62 Laboratories)
Dog
Horse, Human, Mink, Mouse
Hamster
Mouse, Human, Marmoset, Rat
Mongoose
Human
Human
Gibbon
Mink
Human
Monkey
Horse, Human
Mouse
Human
Rabbit
Dog
Rat
Chicken, Human, Mink, Monkey
Nelson-Rees, WA, et al. Science 212,446, 1981
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1980 – 2003 Interspecies and Intraspecies
cross-contamination
Cellular cross-contamination
Year
No.
%
Type of
contam.
Technology
Reference
1984
275
35%
Interspecies
Karyotyping
Hukku, B. et al. Eukaryotic cell
culture. Plenum Press, 1984
1999
252
18%
Intraspecies
STR profiling
Drexler, HG et al. Leukemia
13:1999.
2003
550
15%
Intraspecies
STR profiling
Drexler, HG et al. Leukemia
17:2003
“Less than 50% of researchers regularly verify the identities of their cell
lines using standard methods such as DNA fingerprinting by STR
analysis”
Buehring, G.C., et al. (2004) In Vitro Cell Dev Biol 40:211
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2004 – 2010: Cellular cross-contamination
persists …
Year
9
Title of article
Reference
2004
LCC15-MB cells are MDA-MB-435: a review of
misidentified breast and prostate cell lines..
Clin Exp Metastasis. 21(6):535,
2004.
2007
MDA-MB-435: The Questionable Use of a
Melanoma Cell Line as a Model for Human
Breast Cancer is Ongoing
Cancer Biology & Therapy 6:9,
1355, 2007.
2008
Deoxyribonucleic Acid Profiling Analysis of 40
Human Thyroid Cancer Cell Lines Reveals
Cross-Contamination Resulting in Cell Line
Redundancy and Misidentification.
J Clin Endocrinol Metab.
93(11):4331, 2008.
2009
Genetic Profiling Reveals Cross-Contamination
and Misidentification of 6 Adenoid Cystic
Carcinoma Cell Lines: ACC2, ACC3, ACCM,
ACCNS, ACCS and CAC2.
PLoS one. 4(6):e6040, 2009
2010
Verification and Unmasking of Widely Used
Human Esophageal Adenocarcinoma Cell
Lines.
JNCI. 102(4):271, 2010
Impact of cellular contamination on research
Misidentification of frequently used esophageal adenocarcinoma cell lines
(EAC)
Purported
STR confirmed (ATCC STRProfile database)
SEG-1
Esophageal
adenocarcinoma cell line
H460 (ATCC® HTB-177™)
Lung carcinoma (large
cell lung cancer)
BIC-1
Esophageal
adenocarcinoma cell line
SW620 (ATCC® CCL-227™)
Colorectal
adenocarcinoma
SK-GT-5
Esophageal
adenocarcinoma cell line
SK-GT-2
Gastric fundus
carcinoma
Experimental results based on contaminated cell lines …
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Clinical trail recruiting EAC patients
100 scientific publications
At least 3 NIH cancer research grants
11 US patents
Boonstra, J.J., et al. (2010) JNCI.102(4):271
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Consequences of cellular contamination
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Loss of cell line
Loss of time and money
Misinformation in the public domain
Discordant or irreproducible results
Private embarrassment /public humiliation
The problem of misidentified cell lines
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Misidentified cell lines use is widespread
Problem not readily recognized by …
• scientists
• reviewers of journals
• editors
• funding agencies
Institutionalized ignorance; apathy
“Cases of Mistaken Identity”
“For decades, biologists working with contaminated or misidentified cell
lines have wasted time and money and produced spurious results;
journals and funding agencies say it’s not their job to solve this
problem”
Rhitu Chatterjee. Cases of Mistaken Identity (2007) Science
15:928
Response:
“It is hard for me to fathom that the researchers themselves are willing
to ignore this risk (misidentified cell line) that jeopardizes their work and
are not themselves screaming for ways to ensure that they have pure
cell lines for their research.”
Michael T. Hamilton, Fire/Rescue Battalion, Chief, Montgomery
County Fire and Rescue Service (MCFRS). Science online 2007.
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2007: Eradication of cross-contaminated cell
lines: call for action
Stakeholders have a responsibility to prevent and reduce use of
misidentified cell lines
Nardone, R. (2007) Cell Biol Toxicol 23:367.
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2007: Open Letter by Roland Nardone
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Open letter to Michael Leavitt; Secretary of US Dept. Health
Response by NIH: NIH Notice Number: NOT-OD-08-017, Nov. 28,
2007 – encourages cell line authentication
http://grants.nih.gov/grants/guide/notice-files/NOT-OD-08-017.html
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Editors of journals are requesting
authentication of cell lines
…as a prerequisite for publication
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2009: Establishment of an international
consensus standard for authentication of
human cell lines
ASN-0002 - Authentication of Human Cell Lines:
Standardization of STR Profiling
January 25, 2012: Final action by ANSI
February 2, 2012: Published date
Chaired by John RW Masters, University College of London and
Co-chaired by Yvonne A. Reid, ATCC
Barallon, R. et al. (2010) In Vitro Cell Dev Biol Anim 46:727
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ASN-0002 - Authentication of human cell lines:
standardization of STR profiling
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The standard describes a consistent, inexpensive and
universally applicable method for authenticating new and
established cell lines and their criteria for use.
Section of the standard is modeled after the Scientific
Working Group on DNA Analysis Methods (SWGDAM)
interpretation guidelines of the forensic community.
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Peak amplitude
Use of controls
Allele designation
Data interpretation
STR database as part of the NCBI BioSample Database; to
contain registered cell lines with STR profiles (under
development).
Misidentification of cell lines
“Evidence suggests that up to a third of established tumour cell lines
being used in scientific and medical research is affected by inter- or
intra-species cross-contamination, or have been wrongly identified,
thereby rendering many of the conclusions doubtful if not completely
invalid.”
Lancet Oncology, Contamination of cell lines – a conspiracy of silence Vol.
2, July 2001, p. 393
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STR profiling for speciation and detecting
cellular cross-contamination
Intraspecies identification (within species; human)
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STR analysis: variation in the number of tandem repeats
HLA typing: variation in human leukocyte antigen gene
SNP analysis: variation in single nucleotide – polymorphism
Interspecies identification (between species)
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Isoenzyme analysis: post-translational modification of enzymes
COI analysis: amplification of mitochondrial cytochrome C oxidase I
gene
Karyotyping: differences in metaphase chromosome numbers for each
species
©2011 American Type Culture Collection (ATCC)
Identification of human cell lines
Technologies
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Power of discrimination
Isoenzyme (G6PD)
2
(fast type A and slow type B)
Karyotyping (G-banding)
100s
HLA typing
1,000s
STR analysis
100,000,000s
©2011 American Type Culture Collection (ATCC)
Short Tandem Repeat (STR) analysis for
intraspecies identification of human cell line
DNA location
Degree of
repetition
Number of loci
Repeat unit length
Satellite DNA
(centromere)
103 to 107
1 to 2
2 to several thousand bp
Minisatellite DNA
(telomere)
2 to several
hundred
Many thousands 9 to 100 bp
Microsatellite DNA
(STRs); randomly
scattered
5 to about a
hundred
104 to 105
1 to 6 bp
STR profiling a method for cell line authentication!
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Properties of STRs for DNA profiling
Power of discrimination 1:1.2 x 10E8
Locus name
Chromosome
location
Repeat motif
No.
repeating
units
No. alleles
observed
D16S539
16q24-gtr
GATA
5-15
10
D7S820
7q11.21-22
GATA
6-15
22
D13S317
13q22-q31
TATC
5-15
14
D5S818
5p21-q31
AGAT
7-16
10
CSF1PO
5q33.3-34
TAGA
6-16
15
TPOX
2p23-pter
GAAT
6-13
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vWA
12p23-pter
[TCTA]
[TCTG]
10-24
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THO1
11p15.5
TCAT
3-14
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Amelogenin
Gender determination (not STR marker)
Butler, J.M. Forensic DNA Typing, 2001
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Advantages of STR analysis
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Target sequence consists of microsatellite DNA
Typically use 1-2 ng DNA
1 to 2 fragments; discrete alleles allow digital record of data
Highly variable within populations; highly informative
Advantages of STR analysis
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Banding pattern is reproducible
PCR amplifiable, high throughput
Small size range allows multiplexing
Allelic ladders simplify interpretation
Small product size compatible with
degraded DNA
Rapid processing is attainable
Outline of STR profiling procedure
Resolve PCR fragments
(Capillary electrophoresis)
Extract DNA
Size PCR Fragments
(GeneScan software)
PCR amplified sample
PowerPlexv1.2 System)
Convert PCR fragment sizes to
alleles
(Genotyper software)
Spot onto FTA® paper
Create reference database
• Curate
• Global comparisons
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STR polymorphism
homozygous
TATC
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heterozygous
TAGA
9,10
Gender is important for identification
(amelogenin gene)
male
AMELX
AMELY
female
AMELX gene contains a 6 bp deletion in the intron 1
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Human cell line identification: STR analysis
2 unrelated cell lines (separate individuals, female in origin)
K562
WS1
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D5S818
D13S317
D7S820
D16S539
vWA
THO1
Amel.
TPOX
CSF1PO
K562
11, 12
8
9, 11
11, 12
16
9.3
X
8, 9
9, 10
WS1
13
12
9, 10
10, 11
17, 18
8, 10
X
8, 9
10, 13
Human cell line identification: STR analysis
2 related cell lines (same individual; male in origin)
HAAE-2
aortic artery
HFAE-2
femoral artery
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D5S818
D13S317
D7S820
D16S539
vWA
THO1
Amel.
TPOX
CSF1PO
HAAE-2
12,13
11,12
8,10
12,13
14,18
7,9
X,Y
10,11
10,11
HFAE-2
12,13
11,12
8,10
12,13
14,18
7,9
X,Y
10,11
10,11
STR analysis used to monitoring genomic
stability
Donor
Token
(Pre-MCB)
Seed
(MCB)
Distribution
(WCB)
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Case study 1: cellular cross-contamination
SK-OV-3
Ovary
SK-OV-3 +
cell line X
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Case study 2: gender misidentification
Human cell line purported to be of female origin
Y paint
STR analysis
G-banding
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Are your cells REALLY what you think they are?
Common sources of cellular contamination
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Getting cell lines from colleague down the hall
Continuous culturing of working cell banks
Use of feeder cells
Mislabeling of culture flasks
Working with multiple cell lines concurrently
Performing STR analysis
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Gene sequencer
Thermocycler
Primer kits from manufactures (e.g., Promega)
STR database of human cell lines
Experienced technicians
Interpreting STR data
Criteria for determining quality STR profile
analysis for reliable and interpretable results
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Validation of procedure
Setting of analytical threshold required for interpretation of
results.
Use of appropriate controls (positive and negative).
Ability to evaluate internal lane size standards and allelic ladders.
Appropriate assignment of allele to appropriate peaks or bands.
Ability to determine appropriate peak height or peak threshold.
Ability to detect artifacts, i.e. stutter peaks, dye blobs, dye pullups, microvariants, off-ladder alleles, etc.
Case study 3: complexities of STR patterns
vWA or THO1?
Off ladder allele
DNA Size (bp)
100 pg
template
LOH or allele
drop-out?
5 pg
template
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Services for STR typing of cell lines
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Cell Banks
Paternity testing labs
Universities
Core labs
ATCC®CRL-2123™, mIMCD-3,
kidney, grown on Matrigel™
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STR testing results
No. of
samples
%
Mixture
4
4
Non-human
2
2
Misidentified
Unique (no match in ATCC
database)
Exact match to expected
4
4
30
31
40
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Similar/related to expected
17
18
97
100
Result
TOTAL
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Steps for reducing cellular contamination
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Good documentation
Highly trained technicians
Good aseptic techniques
Use one reservoir of medium per
cell line
Aliquot stock solutions/reagents
ATCC®HTB-174™, NCI-H441, human
papillary adenocarcinoma differentiated
under air-liquid interface conditions
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Steps for reducing cellular contamination
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Label flasks (name of cell line, passage number, date of
transfer (use barcoded flasks when available)
Work with one cell line at a time in biological safety
cabinet
Clean biological safety cabinet between each cell line
Allow a minimum of 5 minutes between each cell line
ATCC® CCL-2™; Hela, cervical carcinoma. Scanning EM
of cultured HeLa cell undergoing apoptosis.
Steps for reducing cellular contamination
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Quarantine “dirty” cell line from
“clean” cell line
Manageable work load (reduce
accidents)
Clean laboratory (reduce bioburden)
Legible handwriting (printed labels)
IPSC colony, on mouse feeder cells,
derived from ATCC® CCL-65™, turner
syndrome fibroblasts, expressing
OCT4, SOX2, KLF4 and cMYC.
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Steps for reducing cellular contamination
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Monitor for cell line identity and characteristics
contamination, routinely
Use seed stock (create master stocks)
Create “good” working environment
Review and approve laboratory notebook
Obtain cell line from a reputable source
ATCC® CRL-1730™, HUVEC expressing CD34
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THANK YOU
Yvonne A. Reid, PhD
[email protected]
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