Human Cell Line Authentication. Why is it so important?

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Human Cell Line Authentication. Why is it so important? Rosa M. Prieto Head of the UAT (Unitat d’Alta Tecnologia, High Technology Unit) SCTs-VHIR 11/06/2015

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1. Introduction

2. Cell line misidentification

3. Cell line authentication UAT service

1. Introduction

Henrietta Lacks

Died of terminal cervical cancer in 1951

First inmortal human cells ever grown “in vitro”

(without her consent)

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24

24% of all contaminants identified are HeLa cells

1. Introduction

1. Introduction

Do we really know which cell line are using for our experiments?

1. Introduction

1. Introduction

Validity of the scientific results produced using these cell lines?

2. Cell line misidentification

Causes of cell line misidentification:

1. Mislabelling

2. Cross-contamination (human error, selective pressure in culture)

3. Poor culture techniques

4. Lack of standarization in cell naming

A cell line is misidentified when its DNA profile is no longer consistent with the individual donor from whom it was first established.

15-20 % of the human cell lines currently used in medical research are misidentified

2. Cell line misidentification: lack of standarization in cell naming

A resource for cell line authentication, annotation and quality control.

Mamie Yu.et al Nature 520, 1-5 (2015) doi:10.1038/nature14397

Catalogue of synonymous cells lines (derived from the same patient/share the same DNA profile)

2. Cell line misidentification.

2. Cell line misidentification.

Some examples:

37 cell lines

438 cell lines

2. Cell line misidentification.

Cell line authentication aims to compare the test cell line to other samples from the same donor (or against a database).

Methods for cell line authentication:

- Isoenzyme analysis

- Kariotyping

- DNA barcoding (mtDNA)

- DNA fingerprinting

- SNP analysis

3. Cell line authentication. STR profiling.

Standard for authentication of human cell lines by STR profiling developed by the American Type Culture Collection Standards Development Organization, Workgroup ASN-0002 (2011)

Approved by the American National Standards Institute (ANSI)

3. Cell line authentication. STR profiling.

3. Cell line authentication. STR profiling. ANSI/ATCC standards.

STR microsatellite regions: 2-6 bp repeats, distributed across the genome

Hotspots for homologous recombination events

Polymorphism: number of repeats (6-21) betwen alleles/loci in unrelated cell lines

Profile of diploid cell lines consists of two numbers for each loci (number of repeats at each allele)

STR (short tandem repeat) PROFILING

A minimum of 8 STR core loci Gender

2) Compare % match vs. donor or vs. STR profiling database

1) STR DNA profiling.

3. Cell line authentication. STR profiling workflow.

STR (short tandem repeat) PROFILING

GenePrint 10 System (Promega)

Genomic Analyzer System ABI3130XL

GeneMapper v3.7 (Life Technologies)

STR profiles Database (ATCC, DSMZ,

RIKEN, CLIMA...)

Authentication of Human Cell Lines by STR DNA Profiling Analysis Yvonne Reid, PhD, Douglas Storts, PhD, Terry Riss, PhD, and Lisa Minor, PhD. Assay Guidance Manual [Internet].

3. Cell line authentication. STR profiling. ANSI/ATCC standards.

3. Cell line authentication. STR profiling results.

STR PROFILING RESULTS

% Match (80-100): related cell lines (same donor) A small amount of STR profile variation can be observed due to genetic drift with passage, particularly in cell lines with

genetic instability. Variation may also be related to testing methods or data interpretation.

% Match (50-80): further investigation (additional loci, alternative test method-SNPs) % Match (0-50): non-related cell lines (different donors)

3. Cell line authentication. STR profiling results interpretation.

-Kits are developed for HUMAN cell lines.

-STR profiles are useful to establish if two cell lines (problem vs. reference /problem vs. database) are related or unrelated (if they come from the same donor). STR profiling does not provide information about genomic modifications (microarrays, NGS...)

-If two cell lines come from the same donor, STR profiling will not discriminate between them.

-STR profiles can be altered by many type of stresses causing loss of heterozigosity or genomic rearrangements (extensive passaging, contamination with microorganisms, passage through animals, exposure to drugs...). These kind of changes cannot be evaluated by this method.

-The same occurs with unstable cell lines (i.e. cancer), potential mixes of cell lines... whose STR profiles can be difficult to interpretate.

- Detection of contamination of human cell lines with mouse cells is also possible using specifically designed PCR primers .

3. Cell line authentication. STR profiling tips.

Two related cell lines (same

donor)

Two unrelated cell lines (CML

vs. skin fibroblasts)

3. Cell line authentication. STR profiling results interpretation.

Genetic instability: LOH, peak imbalance

3. Cell line authentication. STR profiling results interpretation.

Multiple peaks at multiple loci:

genomic aberrations vs.

Cross-contamination

Authentication of Human Cell Lines by STR DNA Profiling

Analysis. NCBI book.

Nature 520 (2015): 307

Validated source of cell lines/cell line is not in the ICLAC list

3. Cell line authentication. Incorporating authentication into everyday culture practice.

- Cell line OK!

- Cell line NO OK according to STR profiling: Supposedly human cell line is a mouse cell line.

Human cell lines contaminated with mouse cells at different rates depending on the number of passages.

Mutually exchanged cell lines in a single lab.

Two cell lines mixed.

Cells purchased from ATCC, whose profile does not match the ATCC database itself.

http://www.atcc.org/Products/Cells_and_Microorganisms/Cell_Lines/Misidentified_Cell_Lines.aspx

3. Cell line authentication. Things that sometimes happen....

Try to obtain cells from reliable sources (cell culture banks).

Avoid to work with cell lines reported as “problematic”. False cell

lines (misidentified with no authentic stock) should not be used.

Check the identity of each new cell line at its reception and before

making stocks/start working with it.

Authenticate cell lines periodically (*).

Renew periodically “working” cells from master stocks and work

with low passages. http://www.atcc.org/~/media/PDFs/Technical%20Bulletins/tb07.ashx

Avoid suboptimal culture conditions that accentuate genetic drift:

overpassaging/overdilution. Feed cells regularly. Watch cells.

Check mycoplasma contamination periodically (*)

Be careful with mislabelling errors and cross-contamination:

Work in the hood with one cell line at a time

Dedicate medium bottles for use with only a cell line

Be careful with labelling (name/data/passage number)

Avoid aerosol formation when working

3. Cell line authentication. Good laboratory practices.

3. Cell line authentication. Good laboratory practices.

Mycoplasma contamination:

- Very frequent, worldwide (continuous cell lines:15-35%)

- Can go unnoticed due to visible signs of contamination. Cells do not die but mycoplasma alters many cellular functions (growth, morphology, metabolism, antigenicity....) → Results?

- Mycoplasma is not affected by antibiotics and pass through standard 0,22 µm.

- Prevention: be aware of the most common sources of infection and observe good cell culture practice.

- Detection: test all actively growing cell lines at regular intervals.

3. Cell line authentication. Mycoplasma testing.

3. Cell line authentication. Mycoplasma testing.

http://www.nature.com.are.uab.cat/authors/policies/availability.html#further

3. Cell line authentication. Pre-requisite for publication in a growing number of journals.

3. Cell line authentication: pre-requisite for publication in a growing number of journals.

Archivo de

resultados

C=50-100 ng/µl,V≥5 µl

A260/A280 ≈ 1,8

UAT

UAT

HUMAN CELL LINE AUTHENTICATION (PER RESEARCHER/REQUEST) RATE

1-4 SAMPLES 48,20 €

≥5 SAMPLES 45,90 €

3. Cell line authentication. New UAT service.

Bring purified DNA from

cell lines to UAT

Samples will be shipped the first

Monday of every month.

For urgent determinations, please

contact UAT.

Detection of contamination of human cell lines with mouse cells

will be available soon.

3. Cell line authentication. UAT service request.

-Sample name

-Cell line expected ID

Requested data:

Deliverables: -Electropherogram

-Report including %match with the reference cell line/database

Thank you!

www.vhir.org

uat@vhir.org

Ext. 4179

Bibliography and resources

- Match criteria for human cell line authentication. Where do we draw the line? Int. J. Cancer 132 (2013)

- Cell line authentication demystified. Nature Methods 11 (5):2014

- Guidelines for the use of cell lines in biomedical research. BJC 111 (2014)

- A resource for cell line authentication, annotation and quality control. Nature 520 (2015)

- Time to tackle cells’ mistaken identity. Nature 520 (2015)

www.atcc.org www.iclac.org www.dsmz.de