Cell Line Authentication

It has been estimated that ~18-36% of cell lines utilized in biomedical research are contaminated and/or are completely misidentified1. Consequently, verification of human cell line identity is of critical significance since the validity of data obtained depends on authenticity of the cell line. TRIP offers Short Tandem Repeat (STR) analysis to verify the authenticity of human cell lines.  Some funding organizations, along with major publishers, such as those affiliated with the American Association for Cancer Research (AACR), have established requirements for cell line authentication prior to publication.

Data from cell lines submitted by UW investigators in 2017 indicates that: 28% of cell lines submitted did not match the line compared to, 2.5% of cell lines submitted were contaminated and 26.3% of cell lines submitted were a match to a completely different line than expected!

We continue to strive to work with researchers to improve those statistics and in 2019 the percentage of cell lines that didn’t match what they were compared to dropped from 28% to 3.8%. Cell lines that matched to a completely different line dropped from 26.3% to 3.8%. That is an incredible difference!

For questions or inquiries, please contact Rebecca Baus at rmbaus@wisc.edu or trip@pathology.wisc.edu.

For pricing please see our TRIP Fee Schedule. To request STR services, go to our iLab webpage.

Analysis Method:  Analysis of 15 independent human STR microsatellite loci (D3S1358, TH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, CSF1PO, Penta D, vWA, D8S1179, TPOX and FGA) and a gender determination loci (Amelogenin) is conducted using the Promega PowerPlex® 16 HS system, multiplex PCR amplification, capillary electrophoresis (ABI 3500) and GeneMapper (v.4.1) software.

Results:  A report is generated indicating STR and Amelogenin genotypes for all loci. Report includes an interpretative comment regarding any indication of possible sample contamination (2% sensitivity) and evaluation (identification or authentication of cell line identity) relative to a publication, repository database or a baseline specimen.

Sample submission requirements:

  1. DNA (20µL at a [DNA] > 10ng/µL*) *TRIP laboratory will determine DNA concentration [DNA]
  2. Cultured Cells (~ 2,000,000 cells)

1 “The costs of using unauthenticated, over-passaged cell lines:  how much more data do we need?” Hughes, P., et al.  BioTechniques 43: 575-586, 2007.

RESOURCES

The ghosts of HeLa: How cell line misidentification contaminates the scientific literature By Serge P. J. M. Horbach and Willem Halffman

Molecular Capabilities in TRIP by William Rehrauer 2016

Human Cell Line Authentication by William Rehrauer 2018

Authentication of Human Cell Lines by DNA STR Profiling by Gabriela Saldanha 2018

Cell Line Authentication Seminar 2019 – TRIP-Promega

FAQs

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What is the turnaround time?

The STR report is ready within 10 business days.

How do I prepare my sample for Cell Line Authentication?

Samples can be submitted as either DNA or cell pellet. All samples should be in a 1.5mL microfuge tube.

DNA: A minimum concentration of 10ng/µL genomic DNA is required however, around 50ng/µL is ideal. A minimum volume of 20µL is required regardless of concentration. Contact us if you are having difficulty meeting this volume requirement.

Cells: Cells should be counted in order to provide approximately 2 million cells. Counts excessively higher than 2 million will create technical difficulties. Two million cells pelleted will look like <5µL if it were a liquid volume.

Cells should be pelleted and washed. Pipet off as much media/wash buffer as possible leaving only the cell pellet. You may store your cell pellet at 4°Celcius until you are ready to drop them off. Cells may be transported in a biohazard bag inside of a secondary container at room temperature. Contact us if you are having difficulty meeting the cell count requirement. This is only an estimated count and fewer cells will be feasible.

What are STRs?

Short Tandem Repeats, or STRs, are repeats in our DNA sequence that are 1-6 base pairs in length, generally 5-50 times. Because of the variation of repeat numbers between individuals, these are useful for identification. Much like forensic DNA typing of evidence at a crime scene used to compare profiles of individuals, we can use STRs to create a genetic profile for cell lines allowing us to compare to a known profile and “authenticate” the line.

How often should I authenticate my cell lines?

-Upon receiving a cell line from another source to confirm identity and establish a baseline
-Prior to freezing
-Every other month while growing in culture
-Prior to publication

Dunham JH and Guthmiller P. Doing Good Science: Authenticating Cell Line Identity. [Internet] 2012. Available from: http://www.promega.com/resources/pubhub/cell-line-authentication-with-strs-2012-update/

Do you authenticate non-human cell lines?

We only perform Human Cell Line Authentication. The Promega PowerPlex ® 16 HS kit that we use consists of 15 human STR markers in addition to the sex determinant marker, Amelogenin.

What will this service provide for me?

When performing Cell Line Authentication through the TRIP lab we will provide you with a report detailing the genetic profile of your cell line. This genetic profile will be compared to a known profile, when available, detail any signs of ploidy, allelic imbalance, microvariants, or contamination. Your initial baseline submissions will be kept on file to compare to future samples. You may request a copy of the electropherogram at any time.

This report is put together by TRIP personnel who have authenticated over 3500 samples since we began this service in 2014. It is affirmed by William Rehrauer, Ph.D., co-Director of TRIP – Molecular and Director of the Clinical Molecular Diagnostics lab at the University of Wisconsin Hospital and Clinics.

If, at any time, you have questions about your results we are available to answer questions via email or appointment.

What if my cell line doesn’t match the comparison profile 100%?

Standards for cell line authentication have been promoted with suggested algorithms to determine degrees of “relatedness” and have been recommended as a way to interpret results from STR profiling of human cell lines. In general, >80% allelic match is common between related samples, whereas < 50% allelic match is unrelated (Capes-Davis A, et al., Match criteria for human cell line authentication: Where do we draw the line? Int. J. Cancer. 132, 2510-2519. 2013).

Do I have to authenticate my cell lines?

Many journals request cell lines to be authenticated prior to publication. This list is continually growing as the scientific community becomes more aware of the effects misidentification can have on research.

AACR Journals:

Cancer Discovery
Cancer Research
Clinical Cancer Research
Cancer Epidemiology, Biomarkers & Prevention
Molecular Cancer Research
Molecular Cancer Therapeutics
Cancer Prevention Research
Carcinogenesis
Cell Biochemistry and Biophysics
Cell Biology International

Endocrine Society Journals:

Endocrinology
Endocrine Reviews
Journal of Clinical Endocrinology & Metabolism
Molecular Endocrinology
Hormones and Cancer
International Journal of Cancer
In Vitro Cellular & Developmental Biology – Animal
Journal of Molecular Biology
Journal of the National Cancer Institute
Molecular Vision

Nature Publishing Group:

Nature
Nature Reviews Molecular Cell Biology
Nature Genetics
Nature Reviews Immunology
Nature Reviews Cancer
Nature Reviews Neuroscience
Nature Biotechnology
Nature Methods

Neuro-Oncology

Placenta

PLoS ONE

Society for Endocrinology journals:

Journal of Endocrinology
Journal of Molecular Endocrinology
Endocrine-Related Cancer

What are the chances my cell line is misidentified or contaminated?

Many researchers feel a false sense of security that their cell lines are identified correctly and are clean/not contaminated. TRIP has performed Human Cell Line Authentication on well over 3500 samples since it began this service in 2014. Looking at samples submitted to TRIP by Academic Researchers in 2017, we found that 28.8% of submissions were not a 100% allelic match, 2.5% of submissions were contaminated, and 26.3% were misidentified lines. We have continued to see this trend in 2018 with contamination on the rise (13.2% as of September 2018). When a cell line is misidentified months or years of research time and dollars is spent working with a cell line that is not the line you thought it was. This can easily be prevented by regularly submitting your samples for testing.

The good news is, with continued testing of cell lines and labs disposing of misidentified lines these numbers have improved. We found in 2019, samples submitted to TRIP by Academic Researchers had a decreased percentage in those cell lines that did not match 100% (28.8% down to 3.8%) and misidentified lines (26.3% down to 3.8%). We have made great progress in our goal of assisting researchers in putting out reproducible results using authenticated human cell lines.

Do you perform mycoplasma testing?

Currently, we do not perform mycoplasma testing but are looking into this as a service to offer in the future.

What STR markers do you test?

The Promega PowerPlex® HS System tests 13 CODIS STR markers (FGA, TPOX, D8S1179, vWA, CSF1PO, D16S539, D7S820, D13S317, D5S818, D18S51, D21S11, TH01 and D3S1358) along with Penta D and Penta E and the sex determinant marker, Amelogenin.

How do I find a comparison profile?

Commercial vendors will often provide a genetic profile on their website for the lines they sell. For example, ATCC profiles can be found under the “Specifications” tab when viewing a cell line.

Other places to obtain a comparison profile:

Publications
Cellosaurus database:  https://web.expasy.org/cellosaurus/
The lab you received the cells from

How do you obtain a genetic profile from my cell sample?

DNA is extracted from a cell pellet using our automated Promega Maxwell® 16 Instrument along with the Maxwell® 16 LEV Blood DNA kit. Once DNA is obtained and quantified, a multiplex PCR reaction is performed using the PowerPlex® 16 HS System to co-amplify 15 STR loci and the sex determinant marker, Amelogenin, each labeled with fluorescein, TMR, or JOE. The amplicon is then capillary electrophoresed on the ABI 3500xl Genetic Analyzer. GeneMapper software creates an electropherogram that we will analyze in order to establish a genetic profile for you cell line.

What does an electropherogram look like?

 

This electropherogram is divided into 3 channels. The first channel (blue) includes STR loci D3S1358, Th01, D21S11, D18S51 and Penta E labeled with fluorescein (FL). The second channel (green) includes D5S818, D13S317, D7S820, D16S539, CSF1PO and Penta D labeled with JOE (6-carboxy-4’, 5’-dichloro-2’,7’-dimethoxy-fluorescein). The third channel (black) includes STR loci vWA, D8S1179, TPOX, FGA and the sex determinant marker Amelogenin labeled with TMR (carboxy-tetramethylrhodamine).

The GeneMapper software converts the number of repeats at each of the loci into peaks. The allelic ladder (grey stripes) provides a standard of 209 common alleles. Two peaks at a given loci represent a heterozygous genotype while one peak is homozygous.  Each of these repeat numbers are inherited, one maternally and the other paternally. The top number in the small box represents the number of repeats. A decimal in that number would indicate a partial repeat. The number below that is the peak area. This is how we determine allelic imbalance. Two alleles are considered imbalanced when the expression level is discordant. As a guideline we call imbalance when the area of one peak at a given loci is less than 50% of the area of the other. Allelic imbalance could be the result of choromsomal gains, losses, and/or amplification in a cell line.