Authentication of Key Biological
and/or Chemical Resources
Joseph M. Tuscano, M.D.
Professor of Medicine
UC Davis
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Four Areas of Clarification
• Scientific premise
• Scientific rigor
• Relevant Biological
Variables, Such as
Sex
• Authentication of
Key Biological and/or
Chemical Resources
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Authentication of Key Biological and/or Chemical
Resources
• The quality of the resources used to conduct
research is critical to the ability to reproduce
the results. NIH expects that key biological
and/or chemical resources will be regularly
authenticated to ensure their identity and
validity for use in the proposed studies.
• Key biological and/or chemical resources are those that: 1) may
differ from laboratory to laboratory or over time; 2) may have
qualities and/or qualifications that could influence the research
data; and 3) are integral to the proposed research and may or may
not be generated with NIH funds. These include, but are not
limited to, cell lines, specialty chemicals, antibodies and other
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biologics.
Cell Lines
 Critical resource for in vitro and pre-clinical animal
models
 Major implications in terms of generating reliable and
reproducible data
 Flawed data can be propagated into pre-clinical animal
models and even human clinical trials
 Enormous amounts of time and resources can be
wasted
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Cell Lines
The problem:
 Misidentification
– Previous studies have shown the 15-30% of cell lines can be
misidentified
– Typically due to cell line cross contamination
– Mislabeling
 Genetic changes
– Genomic instability
– Selective pressures
– Unknown effects of experimental conditions
Boonstra JJ et al J Natl Cancer Inst 2010;102:271–4.
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Cell Lines
The problem-Cont:
 Infectious contamination
– Mycoplasma ~5-10%
– Viral infections
 Phenotypic Changes
– Cell surface markers are critically important for identification and
as therapeutic targets
– Expression levels can change over time and effect behavior and
reactivity of the cell line
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Cell Lines
What can be done:
 Source
– Ensure the source is reliable. If not validate
– ATCC, DSMZ
 Validation
– STR profiling
– 01/2009 the ATCC SDO assembled a workgroup to work with
NCBI to develop these essential guidelines titled “The
Authentication of Human Cell Lines: Standardization of STR
Profiling”
– Mutation testing-research specific
– Phenotypic profiling
• Even if validated by STR receptor levels can change
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Cell Lines
What can be done:
Cont.
 Minimize passages
– 10-20 mx
 Training
– Likely biggest source of misidentification (my opinion)
– Ensure proper training and supervision
 Mycoplasma testing
 Mutation testing
– General vs research specific (p53, EGFR etc)
 Always use appropriate controls
– Primary tissue if possible
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Cell Line-based Animal Models
 Reliable data is dependent upon the validation of the cell
lines within a given host
– Use appropriate controls
– Appropriate statistical analysis
 Alternatives
– Primary tissue derived xenografts using NSG models
– PDX modes Jackson Laboratory (> 300 models)
– Large animal “models” of human disease*
• UCD Vet School-a valuable resource
*Animal Models for the Study of Human Disease P. Michael Conn
ISBN: 978-0-12-415894-8. 2013 Elsevier Inc
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Antibodies and Other Biologics
 The application of antibodies across all biologic
disciplines—from basic research to diagnostics to
therapeutics—is pervasive
 Generally devoid of any kind of consistency or
standardization
 Up to the investigators, granting agencies and publishers
to require standardization and/or validation
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Antibodies and Other Biologics
Improving Reproducibility
 Use a manufacturer with hybridoma clone identification,
lot number and appropriate references
 Using laboratory derived antibodies and reagents
– Access to all researchers for independent study and validation
– Required by many publishers, and granting agencies
 Use appropriate positive and negative controls
 Validate results using complimentary methods
– PCR, IHC, IVH for western/immunoblots etc
 Consistent use protocols
– www.nature.com/protocolexchange
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Reducing our Irreducibility
Nature 2013 Checklist
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Reducing our Irreducibility
Nature 2013 Checklist
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RPG Application and Review
Element of Rigor
Section of
Application
Scientific Premise
Scientific Rigor
Research
Strategy
Consideration of Sex
and Other Relevant
Biological Variables
Authentication of Key
Biological and/or
Chemical Resources
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New
Attachment
Criterion
Score
Additional
Contribute
Review
to Overall
Consideration Impact?
Significance
NA
Yes
Approach
NA
Yes
Approach
NA
Yes
NA
Acceptable or
unacceptable
No
Thank You!
Questions?
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