Preventing Culture Shocks:
Fit-For-Purpose Cell Supply
Jim Cooper
ELRIG Drug Discovery, Manchester, 4th September 2013
Organisational structure
Since April 2013:
Culture Collections’ Scientific
Development Group (SDG)
2
Cell Lines are Key Reagents in
the Drug Discovery Workflow
3
Cell Lines are Critical Reagents
PSN1 – 94060601 Human pancreatic adenocarcinoma cell line
4
•
They should be as
physiologically relevant as
possible
•
Their biology and relevance
should be understood
•
They should be verified
•
The supply should be
standard and consistent
•
Robust Quality Control is
paramount
•
New technologies give new
insights into cell QC
Factors Impacting Cell Supply
 Inventory management
 Mycoplasma and other microbial contamination
 Maintenance of desired cell characteristics:
• Finite / primary cell lines
• Over-passaging
• Sub-optimal cryopreservation and storage
• Affect of cell environment on phenotype and selective pressure
 Misidentified or cross contaminated cell lines:
• Awareness
• Consensus
• Test Limitations
5
Inventory Management:
The Challenge of Managing
Legacy in a Cell Repository
What’s really in the freezer?
6
?
7
>95% Accuracy
24% Accuracy
8
2003 – 2008: ECACC LN2 StockCheck
• >40 year’s worth of master,
•
•
•
•
•
9
working, distribution and legacy
stock
>50,000 cell lines
2 x WTE for 5 years
Over 1 million vial locations in 32
consolidated tanks
Inventory software input by
barcode, keyboard and voice
activated interface.
2-3 operation verification of each
location.
10
11
Effect of Sub-optimal
Storage
Plating Profile of Cells Stored at
Client’s Lab
Plating Profile of cells stored at
ECACC
12
Sub-optimal Cryopreservation and
Storage
Poor freezing may have a similar effect as re-cloning or extensive passage.
Effective QC
• Apoptosis
• Plating
• Growth
13
14
15
Mycoplasma Contamination
In 20% of cultures
received by ECACC
Surface of a Vero Cell
under scanning electron
microscopy
16
<Approx 1 m>
Tests for Mycoplasma
ECACC uses:




Culture Isolation (Reference Method)
PCR
Indirect DNA Stain (Hoechst)
Working towards MycoAlert
Regulatory authorities currently only recognise Culture Isolation and
Indirect DNA Stain
17
% accuracy
Accuracy
to
t3
t3
cells
18
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Culture Collections - a PHE Biological Resource
Nested PCR
assays
in house
PCR 2
in house
PCR 1
MycoAlert
hoechst
stain
isolation
90
80
70
60
50
40
30
20
10
0
Over Passaging of Cells and
Prolonged Culture
X
19
20
20
Culture Collections - a PHE Biological Resource
A549 Lamellar bodies: 1976
MUC5A
2013
ABCA3
2D
21
MUC5B
3D
2D
3D
Impact of Cell Environment on
Phenotype:
22
A549 Human Lung Carcinoma - gene expression
is significantly altered with different substrates.
23
Misidentified Cell lines
1952 – HeLa Cell Line
24
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Cell Line
Claimed origin:
C16
Human foetal lung
Chang Liver
Human liver
Clone 1-5c-4
Human conjunctiva
D98/AH2 Clone B
Somatic cell hybrid (HeLa cells and mouse
3T3 cells)
FL
Human amnion
GIRARDI HEART
Human heart
Hep-2C
Human larynx
Hep2
Human larynx
HSG
Human sub-mandibular gland
INT 407
Human embryonic intestine
JIII
Human monocytic leukaemia
KB
Human oral epidermis
L-41
Human bone marrow
L132
Human embryonic lung
WISH
Human amnion
WKD
Human conjunctiva
WRL 68
Human embryonic liver
1970’s
Issue of HeLa
contamination identified
Today:
It’s not just HeLa…
The issue is getting worse
Capes-Davies and Freshney - Comprehensive list of ~400 misidentified cell lines identified in peer reviewed literature
20% of human cancer research has been carried out on
the wrong cell lines
THOUSANDS of recent publications are invalid!
1. MacLeod et al., (1999))
2. Drexler HG et al. (2003.
26
27
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GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
2, 4
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
3
1, 3
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
5
STR profiling
29
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
GATA-GATA
2, 3
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International Cell Line
Authentication Committee
(ICLAC)
ICLAC
International
Cell Line
Authentication
Committee
31
Primer Sets
FSS
FSS SGM
SGM+
D8
D21
Powerplex 1.2
Codis set
Powerplex 16
identifiler+
D8
D8
D8
D8
D21
D21
D21
D21
D7
D7
D7
D7
CSF1PO
CSF1PO
CSF1PO
CSF1PO
D3
D3
D3
THO1
THO1
THO1
THO1
D13
D13
D13
D13
D16
D16
D3
THO1
THO1
THO1
D16
vWA
vWA
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D2
D2
D19
D19
vWA
D18
D18
Am
Am
FGA
D16
FGA
vWA
vWA
vWA
vWA
TPOX
TPOX
TPOX
TPOX
D18
D18
D18
Am
Am
Am
Am
D5
D5
D5
D5
FGA
FGA
FGA
FES
Penta D
F13A1
Penta E
Standardisation of results and guideline
interpretation - STR Profile Match Criteria
•Compare with “standard” profiles on online databases and
profiles in your data set.
•Focus on the 8 Core Alleles and Gender
•Calculate the % match
•
•
•
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ANSI/ATCC ASN-002 Standard (2011)
International Cell Line Authentication Committee (ICLAC) Resources
http://standards.atcc.org/kwspub/home/the_international_cell_line_authentication_committeeiclac_/
Categorisation
•Unique cell line: STR profile “80% or more” unique – no
matches can be found on public access databases
•Authentic cell line: STR profile matches a cell line of the same
designation (same donor) with 80% or more accuracy
•Misidentified or cross contaminated cell line: matches a cell
line of another designation (another donor) with 80% or more
accuracy OR does not match the designated cell line profile (less
than 55% match)
•Cell line is genetically unstable (mutation) : between 56% and
80% match – requires more investigation. (Rare).
34
Example:
Locus
D5S818
D13S317
D7S820
D16S539
vWA
THO1
Amelogenin
TPOX
CSF1PO
Test Sample
Score
12
12
10
8,9
15,16
8
X
10,12
12,13
10,13
9
10,12
11
17
7
X
11
12,14
Total Alleles in Test Sample
13
Total Alleles in Reference Sample
12
Shared Alleles in Test and Reference Samples
4
Percent Match =
Number of Shared Alleles x 2
Total Alleles in Test Sample + Total alleles in the Reference Sample
= 32% Match
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Reference Sample
ICLAC maintains the database of ~400
misidentified cell lines
Freshney R. I. et al (2010) Int J Cancer. 126: 302-04
New cases since ICLAC inception (2012)
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Reference / cell line
Outcome (to date)
4 x Naso-pharangeal cell lines (CNE1, 2 & HNE 1, 2)
fingerprinted as 60-80% match with HeLa
Discussion ongoing. Are they simple cross
contaminations or hybrids?
NCI-H929 Human Myeloma. Breitkopf et al in PNAS
(USA) 2012 claim the cell line expresses a rare BCRABL tyrosine kinase. Fingerprinting showed their NCIH929 cells were actually the leukaemeia cell line: K562.
Discussion followed up through letters to PNAS from
Macleod et al (2012) and a reply from Breitkopf et al
who still claim their NCI-H929 is genuine but has
“chameleon characteristics”.
SLK human Kaposi sarcoma demonstrated to be Caki
(renal cell carcinoma) by Sturzl et al In J Cancer 2012
Added to the database
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Reference / cell line
Outcome (to date)
KU7, used as a human bladder cell line for decades demonstrated as being
Hela by Jager et al, J Urol 2013
Added to database
R06E – a new fruit bat cell line (2009) permissive to vaccinia Ankara shown
by the originating authors (Jordan et al) to be Vero (African Green Monkey)
(2012)
Added to database
1205Lu Melanoma. Claimed by Nair et al to be cross contaminated with
mouse cells (cell line was grown as a xenograft).
Under investigation - the issue
may only relate to one supplier.
Thyroid Medullary Carcinomas RO-H85-1 and RD-81-1 found to be 647-V
bladder carcinoma and HT-29 colon carcinoma respectively
Added to database
Novel MAI cell line (MALT lymphoma), Kuo et al Genes, Chrom and Cancer
2011. The author published the STR profile as part of the cell line
characterisation but did not check it against a database. The paper passed
peer review but it is clear that the profile matches the already established
lymphoma cell line “Pfeiffer”. A quick literature search demonstrated the
author published work on Pfeiffer two years previously.
A letter from ICLAC has been
submitted to Genes
Chromosomes and Cancer.
T1 novel neural stem cell line, Wu et al 2011. The author published the STR
profile as part of the cell line characterisation but did not check it against a
database. The paper passed peer review but it is clear that the profile
matches Hela.
Challenge ongoing
Residual Issues:
Cell Lines with the
same name
No. of Loci
Cell Line
Tumour
Testing Lab
Locus
D5S818
D13S317
D7S820
D16S539
vWA
THO1
Amelogenin
TPOX
CSF1PO
D8S1179
D21S11
D3S1358
PentaE
PentaD
D18S51
FGA
9 loci
T406*
Oral SCC
Zhao 2011
Score
12
12
10
8,9
15,16
8
X
10,12
12,13
16 loci
T406
Glioblastoma
CLS
10,13
9
10,12
11
17
7
X
11
12,14
14
28,30
14,16
7,10
11
13,18
23,26
* Reported identical to seven other SCC cell lines by Zhao in 2011.
Entered into the database of misidentified cell lines
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9 Loci aren’t always adequate…
No. Loci
Testing Lab
Cell Line
Tumour
Locus
D5S818
D13S317
D7S820
D16S539
vWA
THO1
Amelogenin
TPOX
CSF1PO
D8S1179
D21S11
D3S1358
PentaE
PentaD
D18S51
FGA
39
9 loci
ATCC
Cess
Lymphoma
Score
11,12
12
10,12
12
16,17
7,9.3
X,Y
8,9
10,11
9 loci
16 loci
16 loci
DSMZ
ECACC
ECACC
IGR 37 / IGR 39 IGR 39
IGR 37
Melanoma (Same Patient)
16 loci
Castella
LN235
Glioma
11,12
12
10,11
11
17,21
9
X,Y
8,11
11,12
11,12
9
10,12
11
17
7,9
X,Y
8
11,12
14,15
29,32.2
15
14,15
11,12
13,16
22
11,12
12
10,11
11
17,21
9
X,Y
8,11
11,12
14,15
31.2,32.2
17,18
12,16
9
15
22
11,12
12
10,11
11
17,21
9
X,Y
8,11
11,12
14,15
31.2,32.2
17,18
12,16
9
14,15
22
In the absence of
regulation it is up to all
of us to ensure we’re
working with verified
materials and therefore
carrying out valid
research.
40
Uniquely Identifying Non-Human
Cell Lines…
41
Factors Impacting Cell Supply
 Inventory management
 Mycoplasma and other microbial contamination
 Maintenance of desired cell characteristics:
• Finite / primary cell lines
• Over-passaging
• Sub-optimal cryopreservation and storage
• Affect of cell environment on phenotype and selective pressure
 Misidentified or cross contaminated cell lines:
• Awareness
• Consensus
• Test Limitations
42
Thanks
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•
To you all for your attention
•
•
•
•
•
•
•
Steve Grigsby and the Culture Collections’ Logistics Team
Dr Ed Burnett and Dr Liz Penn and rest of the SDG Team
Dr Karen Kempsell and Muhammad Abdulatif, Diagnostic Technologies, PHE Porton
Howard Tolley, Microbial Imaging, PHE Porton
Dr Amanda Capes-Davis and the ICLAC Team
Jamie Taylor and Claire Wilson, Culture Collections’ QC
Diane Fellows, ECACC Operations
•
And…… to my Mother in Law for the frozen red cabbage.
Jim Cooper Cell Biology Applications Scientist
[email protected]
www.phe-culturecollections.org.uk
ELRIG Drug Discovery, Manchester, 4th September 2013