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)
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Cell Lines are Key Reagents in the Drug Discovery Workflow
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Cell Lines are Critical Reagents
PSN1 – 94060601 Human pancreatic adenocarcinoma cell line
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They should be as physiologically relevant as possible
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Their biology and relevance should be understood
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They should be verified
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The supply should be standard and consistent
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Robust Quality Control is paramount
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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
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Inventory Management: The Challenge of Managing Legacy in a Cell Repository What’s really in the freezer?
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?
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>95% Accuracy 24% Accuracy 8
2003 – 2008: ECACC LN2 StockCheck • >40 year’s worth of master, • • • •
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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.
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Effect of Sub-optimal Storage Plating Profile of Cells Stored at Client’s Lab
Plating Profile of cells stored at ECACC
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Sub-optimal Cryopreservation and Storage Poor freezing may have a similar effect as re-cloning or extensive passage. Effective QC
• Apoptosis • Plating • Growth
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Mycoplasma Contamination
In 20% of cultures received by ECACC
Surface of a Vero Cell under scanning electron microscopy 16
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
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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
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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:
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A549 Human Lung Carcinoma - gene expression is significantly altered with different substrates.
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Misidentified Cell lines 1952 – HeLa Cell Line
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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.
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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
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1, 3
GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA GATA-GATA
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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
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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).
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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
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Total Alleles in Reference Sample
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Shared Alleles in Test and Reference Samples
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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 38
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
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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…
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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
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Thanks
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To you all for your attention
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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
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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
