ISO/TS 23511:2023
(Main)Biotechnology — General requirements and considerations for cell line authentication
Biotechnology — General requirements and considerations for cell line authentication
This document defines terms related to cell line authentication in the field of biotechnology. It describes the general principles, detection strategies and analytical methods for cell line authentication. It specifies requirements and key considerations for method selection, quality control parameters, data analysis and reporting. This document is applicable to routine inspection of cell lines in culture and in storage in the fields of basic research, translational studies and product manufacturing. It is also applicable to cell line origin validation in academic and industrial laboratories, cell banks and manufacturing sites. It is primarily applicable to mammalian cells, including human cells. This document does not apply to non-animal cells (e.g. microbial contamination, plant cells), nor to cells in complex matrices (e.g. tissues, organs, organoids, plants).
Biotechnologie — Exigences et considérations générales relatives à l'authentification de la lignée cellulaire
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TECHNICAL ISO/TS
SPECIFICATION 23511
First edition
2023-05
Biotechnology — General
requirements and considerations for
cell line authentication
Biotechnologie — Exigences et considérations générales relatives à
l'authentification de la lignée cellulaire
Reference number
ISO/TS 23511:2023(E)
© ISO 2023
---------------------- Page: 1 ----------------------
ISO/TS 23511:2023(E)
COPYRIGHT PROTECTED DOCUMENT
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address below
or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii
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ISO/TS 23511:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles of cell line authentication .4
4.1 General . 4
4.2 Confirmation of cell origin . 4
4.3 Detection of cross-contamination . 4
4.3.1 Detection of cell line inter-species cross-contamination . 4
4.3.2 Detection of cell line intra-species cross-contamination . 5
4.4 Identification of cell line-specific characteristics . 5
4.4.1 Detection of cell line genome heterogeneity . 5
4.4.2 Detection of cellular differentiation . 6
5 Application scenarios of cell line authentication . 6
6 Sample preparation .7
7 Method options for cell line authentication . 7
7.1 General . 7
7.2 DNA-based cell line authentication methods . 7
7.2.1 Short tandem repeat profiling. 7
7.2.2 Single nucleotide polymorphism profiling . 9
7.2.3 DNA barcoding . 9
7.2.4 Multiplex PCR . 10
7.2.5 Whole genome sequencing . 10
7.3 Related methods for cell line identification . 10
8 Authentication method selection .11
8.1 General . 11
8.2 Cell origin . 11
8.2.1 Confirmation of cell line origin. 11
8.2.2 Identification of cell line gene mutations . 11
8.2.3 Identification of cell line-specific properties . 11
8.3 Species types of cross-contamination . 11
8.3.1 Cell line inter-species cross-contamination . 11
8.3.2 Cell line intra-species cross-contamination .12
8.4 Cell culture methods . 12
8.4.1 Authentication for co-cultured cells .12
8.4.2 Authentication for ex vivo cell culture .12
8.4.3 Authentication for laboratory operation .12
8.5 Authentication purpose . 12
9 Quality control .13
9.1 Operator training . 13
9.2 Instruments and equipment .13
9.3 Reagents . 14
9.4 Validation and verification of methods . 14
9.4.1 General . 14
9.4.2 Validation . 14
9.4.3 Verification . 14
10 Report .15
10.1 Reporting . 15
10.2 Evaluation of measurement uncertainty . 15
iii
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ISO/TS 23511:2023(E)
Annex A (informative) Detection methods for cell line authentication .16
Bibliography .18
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ISO/TS 23511:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
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ISO/TS 23511:2023(E)
Introduction
Cell line authentication is a critical quality control (QC) procedure, which aims to verify a cell line’s
identity and show that it is free of contamination from other cell lines. It has been estimated that a
considerable proportion of the cell lines stored in the United States, Europe and Asia are misidentified
or cross-contaminated, which results in potentially misleading or non-repeatable data, causing
[13]
tremendous waste of time and effort. To facilitate proper utilization of a cell line, the standardization
of procedures used for cell line authentication is urgently needed. This document elaborates on general
requirements for cell line authentication based on the existing national standards and state-of-the-art
methods, aiming to represent and provide guidance to stakeholders in life science, biomedicine and
other related fields.
vi
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TECHNICAL SPECIFICATION ISO/TS 23511:2023(E)
Biotechnology — General requirements and considerations
for cell line authentication
1 Scope
This document defines terms related to cell line authentication in the field of biotechnology. It describes
the general principles, detection strategies and analytical methods for cell line authentication. It
specifies requirements and key considerations for method selection, quality control parameters, data
analysis and reporting.
This document is applicable to routine inspection of cell lines in culture and in storage in the fields of
basic research, translational studies and product manufacturing. It is also applicable to cell line origin
validation in academic and industrial laboratories, cell banks and manufacturing sites. It is primarily
applicable to mammalian cells, including human cells.
This document does not apply to non-animal cells (e.g. microbial contamination, plant cells), nor to cells
in complex matrices (e.g. tissues, organs, organoids, plants).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
cell bank
collection of appropriate containers, whose contents are of uniform composition, stored under defined
conditions, and where each container represents an aliquot of a single pool of cells
[14]
[SOURCE: ICH Q5D ]
3.2
cell line
defined population of cells that has been passaged from a primary culture, and can be maintained in
culture for an extended period of time, retaining stability of certain initial phenotypes and functions
for its intended use
Note 1 to entry: A primary culture is a culture started from cells, tissues or organs taken directly from an
organism, and before the first subculture, propagation and consecutive passages in vitro.
1
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ISO/TS 23511:2023(E)
3.3
cell line authentication
process by which the cell line (3.2) identity is verified and shown to be free of contamination from other
cell lines
3.4
cell line identification
process by which the cell line (3.2) identity is verified, which includes confirmation of cell origin, and
identification of cell-specific characteristics
3.5
microbial contamination
presence of exogenous:
a) bacteria and/or fungi;
b) viruses; and/or
c) foreign inter- or intra-species cell lines (3.2) in a cell culture
Note 1 to entry: Some cell lines have endogenous virus/viral sequences.
Note 2 to entry: Point c) is commonly known as “cell cross-contamination”.
3.6
detection limit
lowest quantity of a substance that can be distinguished from the absence of that substance with a
stated confidence limit
[SOURCE: ISO 14687:2019, 3.5]
3.7
DNA barcoding
taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to
a particular species
3.8
immunofluorescence
method for studying the distribution of specific protein antigens in cells by combining immunological
methods (antigen-specific binding) with fluorescent labelling techniques
3.9
cell line inter-species cross-contamination
contamination of a cell culture by cells derived from different species
3.10
cell line intra-species cross-contamination
contamination of a cell culture by the same type of cells (from different individuals) or different types
of cells (from the same or different individuals) derived from the same species
3.11
isozyme analysis
isoenzyme analysis
separation technique based on electrophoresis to generate patterns of enzymatically active
polypeptides with identical specificity (3.19) but of different molecular structure
3.12
karyotype analysis
chromosomal analysis in each cell to detect aneuploidy, structural abnormalities and ploidy
2
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ISO/TS 23511:2023(E)
3.13
MPS
massively parallel sequencing
sequencing technique based on the determination of incremental template based polymerization of
many independent DNA molecules simultaneously
Note 1 to entry: Massively parallel sequencing technology can provide millions or billions of short reads per run.
[SOURCE: ISO 20397-2:2021, 3.30]
3.14
cell line misidentification
incidence where the cell line (3.2) identity is incorrectly given through mislabelling
3.15
PCR
polymerase chain reaction
enzymatic procedure which allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005, 3.4.1]
3.16
sensitivity
quotient of the change in an indication of a measuring system and the corresponding change in a value
of a quantity being changed
[SOURCE: ISO/IEC Guide 99:2007, 4.12, modified ― Preferred term “sensitivity of a measuring system”
and notes to entry deleted. “changed” replaced “measured”.]
3.17
STR
short tandem repeat
variable segments of DNA that are composed of multiple adjacent two to five basepair long sequences
3.18
SNP
single nucleotide polymorphism
single nucleotide variation in a genetic sequence that occurs at an appreciable frequency in the
population
[SOURCE: ISO 25720:2009, 4.23]
3.19
specificity
property of a method to respond exclusively to the characteristic or analyte under investigation
[SOURCE: ISO 24276:2006, 3.1.4]
3.20
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified ― Notes to entry deleted.]
3.21
WGS
whole genome sequencing
methods that approach determination of the entire nucleotide sequence of the nuclear DNA of eukaryotic
organisms
3
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ISO/TS 23511:2023(E)
3.22
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified ― Notes to entry deleted.]
4 Principles of cell line authentication
4.1 General
Multiple test methods that rely on genomic analysis combined with phenotypic characteristics can be
used as part of the process of cell line authentication. Purposes of genomic analysis include:
a) confirmation of cell origin;
b) examination of cell species, to ensure that no cell line inter- or intra-species cross-contamination
exists in cell cultures;
c) either identification or confirmation, or both, of certain cell line-specific characteristics. Cell line-
specific characteristics such as gene mutations can be useful supporting evidence for the cell line
authentication. However, many “cell-specific” characteristics are related to tissue type or disease
status and are not unique.
4.2 Confirmation of cell origin
For a newly established cell line, a liquid or solid tissue sample from which a cell line is derived, or
a liquid or solid tissue sample from the same donor from whom the cell line was derived, should be
stored for origin confirmation. The baseline DNA profile of the original sample should be used in cell
line authentication by comparing it to the DNA profiles of subsequent passages. If the source tissue or
blood, or both, are not available, the DNA profile of an early passage stock can be used as the baseline.
DNA-based profiling methods intended for routine genotype analysis include:
a) short tandem repeat (STR) analysis with polymerase chain reaction (PCR) assays followed by
fragment size analysis or by Sanger sequencing;
b) single nucleotide polymorphism (SNP) analysis by single-base extension assay or SNP genotyping
qPCR assays;
c) latest DNA profiling technologies, such as massively parallel sequencing (MPS).
SNP databases of targeted panels are now available for analysis. However, there are no central databases
or universally accepted SNP markers, so any SNP comparison shall be in-house or have similar usage
limitations. The whole genome sequencing (WGS) data of newly or already established cell lines should
be provided as a further information source.
4.3 Detection of cross-contamination
4.3.1 Detection of cell line inter-species cross-contamination
4.3.1.1 Cell line inter-species cross-contamination occurs when a cell line is contaminated by
undesired cells from different species. Cell lines derived from different species have different
characteristics, not all of which are suitable for authentication.
Cell line authentication should be performed with consideration for various characteristics, including:
a) genetic characteristics (e.g. CO1, CytB and ND5 genes);
b) cytogenetic characteristics (e.g. chromosome karyotype, marker chromosome);
4
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ISO/TS 23511:2023(E)
c) biochemical characteristics (e.g. enzyme type);
d) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific
antigens);
e) cell kinetics (e.g. differences in cell division frequency or cell generation time);
f) morphological characteristics (e.g. round, long spindle).
4.3.1.2 Methods based on different measurement principles should be used for the detection of cell
line inter-species cross-contamination. For genetic and cytogenetic characteristics, detection methods
include DNA barcoding, PCR assays and karyotype analysis. DNA barcoding can be used to investigate
the mitochondrial gene sequences associated with species-specific cytochrome c oxidase subunit 1
(CO1) gene. PCR assays utilize either species-specific or degenerate primers, which can amplify DNA
fragments for species identification and can detect lower levels of cross-contamination than Sanger
sequencing-based DNA barcoding. Karyotype analysis can directly reveal cross-contamination by
comparing species-specific chromosomes. Morphological characteristics, cell kinetics, biochemical
characteristics and phenotype are useful to provide supporting data for occurrence of cell cross-
contamination, but are not suitable for authentication testing when used alone.
4.3.2 Detection of cell line intra-species cross-contamination
4.3.2.1 Cell line intra-species cross-contamination occurs when a cell line is contaminated by cells of
the same type (from different individuals) or of different types (from the same or different individuals)
within the same species. Detection of cell line intra-species cross-contamination depends on individual
cell line-specific characteristics, which can include:
a) genetic characteristics (e.g. STR profiling, SNP profiling);
b) genetic sequence (e.g. WGS);
c) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific
antigens);
d) morphological characteristics (e.g. round, long spindle);
e) histology (e.g. extracellular cellular markers).
Morphological characteristics and cell markers are useful to provide supporting data for occurrence of
cell cross-contamination, but are not suitable for authentication testing when used alone.
4.3.2.2 Sequence-specific STR or SNP profiles can be used to discriminate among individuals within
the same species. STR or SNP profiling-based Sanger sequencing or MPS technologies should be used for
detection of cell line identity but they also can provide data about intra-species cross-contamination.
Attention should be given to contamination at an early stage, which can go unnoticed even by these
techniques.
4.4 Identification of cell line-specific characteristics
4.4.1 Detection of cell line genome heterogeneity
4.4.1.1 During extended in vitro cell culturing, cell lines can acquire additional genomic changes
and evolve into multiple genetically, transcriptionally, proteotypically or phenotypically different sub-
clones (e.g. the genetic instability and cell heterogeneity of cancer cell lines).
NOTE 1 Detection of cell line genome heterogeneity is not an authentication test method. Methods for cell line
authentication can be used for detecting cell line genome heterogeneity.
5
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ISO/TS 23511:2023(E)
NOTE 2 It is reported that stocked HeLa cells originated from different laboratories show notable variability
in genome, steady-state mRNA expression, protein expression and protein turnover rates at uniform culture
condition. Moreover, progressive divergence can be observed within a specific HeLa cell line after three months
of continuous culture. The in vitro cell manipulating procedures, such as transfection and gene editing, can also
[15]
lead to genetic heterogeneity.
Detection of genomic changes depends on cell line-specific markers, which can include:
a) genetic sequence (e.g. Sanger sequencing or MPS of DNA);
b) genetic characteristics (e.g. STR profiling, SNP profiling);
c) transcription (e.g. mRNA);
d) karyotype.
4.4.1.2 The detection methods of cell line gene mutations can include high-throughput sequencing
(e.g. WGS) along with karyotype analysis. The COSMIC database provides list of cell line somatic
[16]
mutations found in various human cancers.
4.4.2 Detection of cellular differentiation
4.4.2.1 Both pluripotent and multipotent stem cells can differentiate, either spontaneously or with
external stimuli into certain cell types in vitro. Cellular differentiation within a given cell line can be
detected by various gene expression markers, which can include, but are not limited to:
a) cell surface markers;
b) transcription factors;
c) signalling pathway-related intracellular markers;
d) enzymatic markers.
4.4.2.2 Flow cytometry analysis, immunofluorescent staining and enzyme-linked immunosorbent
assays can be used to detect differentiated cell-specific gene expression, along with gene expression
assays, indicating cellular differentiation status.
NOTE Measurement of cellular differentiation can provide useful supporting data as part of broader cell
line identification but is not suitable for authentication testing when used alone as this relies on genome-based
methods.
5 Application scenarios of cell line authentication
To avoid cell line misidentification and cross-contamination, cell line authentication should be used in
the following scenarios:
a) authentication and characterization of newly established cell lines;
b) routine inspection of cell lines in culture and in stock, especially for rapid growing cell types, cells
in extended culture, cells with unusual phenotype and cells after a selection/sorting process;
c) validation of cell line origin upon receiving from other facilities, before sending the material to
other facilities and prior to banking;
d) validation of a cell line origin after the preparation of a cell bank (i.e. seed stock);
e) authentication of cell lines used in both basic and clinical research when abnormalities were found
in the cultured cells, or after multiple passaging.
6
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ISO/TS 23511:2023(E)
6 Sample preparation
6.1 Samples utilized for cell line authentication can include whole cells, deoxyribonucleic acid (DNA)
and ribonucleic acid (RNA). Identification errors can arise due to errors when labelling or handling
samples, or cross-contamination of cells or DNA
...
© ISO 2023 – All rights reserved
ISO/DTS .2 23511:20222023(E)
Date: 2023-02-13
ISO/TC 276/WG 3
Secretariat: DIN
Biotechnology — General requirements and considerations for
cell line authentication
---------------------- Page: 1 ----------------------
© ISO 2023 – All rights reserved
© ISO 2023
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of
this publication may be reproduced or utilized otherwise in any form or by any means, electronic or
mechanical, including photocopying, or posting on the internet or an intranet, without prior written
permission. Permission can be requested from either ISO at the address below or ISO’s member body in the
country of the requester.
ISO Copyright Office
CP 401 • CH-1214 Vernier, Geneva
Phone: + 41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland.
---------------------- Page: 2 ----------------------
ISO/DTS 23511:2023(E)
Contents
Foreword . v
Introduction. vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles of cell line authentication . 4
4.1 General . 4
4.2 Confirmation of cell origin . 4
4.3 Detection of cross-contamination . 5
4.3.1 Detection of cell line inter-species cross-contamination . 5
4.3.2 Detection of cell line intra-species cross-contamination . 5
4.4 Identification of cell-line-specific characteristics . 6
4.4.1 Detection of cell line genome heterogeneity . 6
4.4.2 Detection of cellular differentiation. 7
5 Application scenarios of cell line authentication . 7
6 Sample preparation . 7
7 Method options for cell line authentication . 8
7.1 General . 8
7.2 DNA-based cell line authentication methods . 8
7.2.1 Short tandem repeat profiling . 8
7.2.2 Single nucleotide polymorphism profiling . 10
7.2.3 DNA barcoding . 10
7.2.4 Multiplex PCR . 11
7.2.5 Whole genome sequencing . 11
7.3 Related methods for cell line identification . 11
8 Authentication method selection . 12
8.1 General . 12
8.2 Cell origin . 12
8.2.1 Confirmation of cell line origin . 12
8.2.2 Identification of cell line gene mutations . 12
8.2.3 Identification of cell-line-specific properties . 12
8.3 Species types of cross-contamination . 12
8.3.1 Cell line inter-species cross-contamination . 12
8.3.2 Cell line intra-species cross-contamination . 13
8.4 Cell culture methods . 13
8.4.1 Authentication for co-cultured cells . 13
8.4.2 Authentication for ex vivo cell culture . 13
8.4.3 Authentication for laboratory operation . 13
8.5 Authentication purpose . 13
9 Quality control . 14
9.1 Operator training . 14
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ISO/DTS 23511:2023(E)
9.2 Instruments and equipment . 14
9.3 Reagents . 15
9.4 Validation and verification of methods . 15
9.4.1 General . 15
9.4.2 Validation . 15
9.4.3 Verification . 16
10 Report . 16
10.1 Reporting . 16
10.2 Evaluation of measurement uncertainty . 16
Annex A (informative) Detection methods for cell line authentication . 17
Bibliography . 19
iv © ISO 2023 – All rights reserved
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ISO/DTS 23511:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work. ISO
collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2. (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of any
patent rights identified during the development of the document will be in the Introduction and/or on
the ISO list of patent declarations received. (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the World
Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
© ISO 2023 – All rights reserved v
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ISO/DTS 23511:2023(E)
Introduction
Cell line authentication is a critical quality control (QC) procedure, which aims to verify a cell line’s
identity and show that it is free of contamination from other cell lines. It has been estimated that a
considerable proportion of the cell lines stored in the United States, Europe and Asia are misidentified or
cross-contaminated, which results in potentially misleading or non-repeatable data, causing tremendous
[1][13]
waste of time and effort. To facilitate proper utilization of a cell line, the standardization of
procedures used for cell line authentication is urgently needed. This document elaborates on general
requirements for cell line authentication based on the existing national standards and state-of-the-art
methods, aiming to represent and provide guidance to stakeholders in life science, biomedicine and other
related fields.
vi © ISO 2023 – All rights reserved
---------------------- Page: 6 ----------------------
TECHNICAL SPECIFICATION ISO/DTS 23511:2023(E)
Biotechnology — General requirements and considerations for cell
line authentication
1 Scope
This document defines terms related to cell line authentication in the field of biotechnology. This
documentIt describes the general principles, detection strategies and analytical methods for cell line
authentication. This document providesIt specifies requirements and key considerations for method
selection, quality control parameters, data analysis and reporting.
This document is applicable to routine inspection of cell lines in culture and in storage in the fields of
basic research, translational studies, and product manufacturing. This documentIt is also applicable to
cell line origin validation in academic and industrial laboratories, cell banks, and manufacturing sites.
This documentIt is primarily applicable for mammalian cells, including human cells.
This document isdoes not intended forapply to non-animal cells (e.g. microbial contamination, plant
cells), nor forto cells in complex matrices (e.g. tissues, organs, organoids, plants).
2 Normative references
The following documents, are referred to in wholethe text in such a way that some or in part, are
normatively referenced inall of their content constitutes requirements of this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated
references, the latest edition of the referenced document (including any amendments) applies.
ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https://www.iso.org/obp
— IEC Electropedia: available at https://www.electropedia.org/
3.1
cell bank
collection of appropriate containers, whose contents are of uniform composition, stored under defined
conditions, and where each container represents an aliquot of a single pool of cells
[14]
[SOURCE: ICH Q5D ]
3.2
cell line
defined population of cells that has been passaged from a primary culture, and can be maintained in
culture for an extended period of time, retaining stability of certain initial phenotypes and functions for
its intended use
Note 1 to entry: A primary culture is a culture started from cells, tissues, or organs taken directly from an organism,
and before the first subculture, propagation and consecutive passages in vitro.
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ISO/DTS 23511:2023(E)
3.3
cell line authentication
process by which the cell line (3.2) identity is verified and shown to be free of contamination from other
cell lines
3.4
cell line identification
process by which the cell line (3.2) identity is verified, which includes confirmation of cell origin, and
identification of cell-specific characteristics
3.5
microbial contamination
presence of exogenous (1) bacteria and/or fungi, (2) viruses, and/or (3) foreign inter- or intra-species
cell lines in a cell culture:
a) bacteria and/or fungi;
b) viruses; and/or
c) foreign inter- or intra-species cell lines (3.2) in a cell culture
Note 1 to entry: Some cell lines have endogenous virus/viral sequences.
Note 2 to entry: Point (3c) is commonly known as “cell cross-contamination”.
3.6
detection limit
lowest quantity of a substance that can be distinguished from the absence of that substance with a stated
confidence limit
[SOURCE: ISO 14687:2019, 3.5]
3.7
DNA barcoding
taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to a
particular species
3.8
examination accuracy
closeness of agreement between an examined value and a true nominal property value of an examinand
3.9
immunofluorescence
method for studying the distribution of specific protein antigens in cells by combining immunological
methods (antigen-specific binding) with fluorescent labelling techniques
3.109
cell line inter-species cross-contamination
contamination of a cell culture by cells derived from different species
3.1110
cell line intra-species cross-contamination
2 © ISO 2023 – All rights reserved
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ISO/DTS 23511:2023(E)
contamination of a cell culture by the same type of cells (from different individuals) or different types of
cells (from the same or different individuals) derived from the same species
3.1211
isozyme analysis
isoenzyme analysis
separation technique based on electrophoresis to generate patterns of enzymatically active polypeptides
with identical specificity (3.19) but of different molecular structure
3.1312
karyotype analysis
chromosomal analysis in each cell to detect aneuploidy, structural abnormalities and ploidy
3.1413
MPS
massively parallel sequencing
sequencing technique based on the determination of incremental template based polymerization of many
independent DNA molecules simultaneously
Note 1 to entry: Massively parallel sequencing technology can provide millions or billions of short reads per run.
[SOURCE: ISO 20397-2:2021, 3.30]
3.15 14
misidentified cell line misidentification
incidence where the cell line (3.2) thatidentity is misidentifiedincorrectly is given through
mislabelingmislabelling
3.1615
PCR
polymerase chain reaction
enzymatic procedure which allows in vitro amplification of DNA
[SOURCE: ISO 22174;:2005, definition 3.4.1]
3.1716
sensitivity
quotient of the change in an indication of a measuring system and the corresponding change in a value of
a quantity being changed
[SOURCE: ISO/ICEIEC Guide 99:2007, 4.12, modified ― All notes and the alternatePreferred term
“sensitivity of a measuring system” have beenand notes to entry deleted, and “measured” was. “changed”
replaced by “changed“measured”.]
3.1817
STR
short tandem repeat
variable segments of DNA that are composed of multiple adjacent two to five basepair long sequences
3.1918
SNP
single nucleotide polymorphism
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ISO/DTS 23511:2023(E)
single nucleotide variation in a genetic sequence that occurs at an appreciable frequency in the
population
[SOURCE: ISO 25720:2009, 4.23]
3.2019
specificity
property of a method to respond exclusively to the characteristic or analyte under investigation
[SOURCE: ISO 24276:2006, 3.1.54]
3.2120
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
[SORCESOURCE: ISO 9000:2015, 3.8.13, modified ― All notes have beenNotes to entry deleted.]
3.2221
WGS
whole genome sequencing
methods that approach determination of the entire nucleotide sequence of the nuclear DNA of eukaryotic
organisms
3.2322
verification
confirmation, through the provision of objective evidence, that specified requirements have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified ― All notes have beenNotes to entry deleted.]
4 Principles of cell line authentication
4.1 General
Multiple test methods that rely on genomic analysis combined with phenotypic characteristics can be
used as part of the process of cell line authentication. Purposes of genomic analysis include:
a) confirmation of cell origin;
b) examination of cell species, to ensure that no cell line inter- or intra-species cross-contamination
exists in cell cultures;
c) identification and/or confirmation of certain cell-line-specific characteristics. Cell-line-specific
characteristics such as gene mutations can be useful supporting evidence for the cell line
authentication. However, many “cell-specific” characteristics are related to tissue type or disease
status and are not unique.
4.2 Confirmation of cell origin
For a newly established cell line, a liquid or solid tissue sample from which a cell line is derived, or a liquid
or solid tissue sample from the same donor from whom the cell line was derived, should be stored for
origin confirmation. The baseline DNA profile of the original sample should be used in cell line
authentication by comparing it to the DNA profiles of subsequent passages. If the source tissue, blood, or
4 © ISO 2023 – All rights reserved
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ISO/DTS 23511:2023(E)
both are not available, the DNA profile of an early passage stock can be used as the baseline. DNA-based
profiling methods intended for routine genotype analysis include:
a) short tandem repeat (STR) analysis with polymerase chain reaction (PCR) assays followed by
fragment size analysis or by Sanger sequencing;
b) single nucleotide polymorphism (SNP) analysis by single-base extension assay or SNP genotyping
qPCR assays;
c) latest DNA profiling technologies, such as massively parallel sequencing (MPS).
SNP databases of targeted panels are now available for analysis. However, there are no central databases
or universally accepted SNP markers, so any SNP comparison needs toshall be in-house or have similar
usage limitations. The whole genome sequencing (WGS) data of newly or already established cell lines
should be provided as a further information source.
4.3 Detection of cross-contamination
4.3.1 Detection of cell line inter-species cross-contamination
4.3.1.1 Cell line inter-species cross-contamination occurs when a cell line is contaminated by undesired
cells from different species. Cell lines derived from different species have different characteristics, not all
of which are suitable for authentication.
Cell line authentication should be performed with consideration for various characteristics, including:
a) genetic characteristics (e.g. COI, CytB and ND5 genes);
b) cytogenetic characteristics (e.g. chromosome karyotype, marker chromosome);
c) biochemical characteristics (e.g. enzyme type);
d) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific antigens);
e) cell kinetics (e.g. differences in cell division frequency or cell generation time);
f) morphological characteristics (e.g. round, long spindle).
4.3.1.2 Methods based on different measurement principles should be used for the detection of cell line
inter-species cross-contamination. For genetic and cytogenetic characteristics, detection methods
include DNA barcoding, PCR assays and karyotype analysis. DNA barcoding can be used to investigate the
mitochondrial gene sequences associated with species-specific cytochrome c oxidase subunit 1 (CO1)
gene. PCR assays utilize either species-specific or degenerate primers, which can amplify DNA fragments
for species identification and can detect lower levels of cross-contamination than Sanger sequencing-
based DNA barcoding. Karyotype analysis can directly reveal cross-contamination by comparing species-
specific chromosomes. Morphological characteristics, cell kinetics, biochemical characteristics, and
phenotype are useful to provide supporting data for occurrence of cell cross-contamination, but are not
suitable for authentication testing when used alone.
4.3.2 Detection of cell line intra-species cross-contamination
4.3.2.1 Cell line intra-species cross-contamination occurs when a cell line is contaminated by cells of the
same type (from different individuals) or of different types (from the same or different individuals)
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ISO/DTS 23511:2023(E)
within the same species. Detection of cell line intra-species cross-contamination depends on individual
cell-line–-specific characteristics, which can include:
a) genetic characteristics (e.g. STR profiling, SNP profiling);
b) genetic sequence (e.g., whole genome sequencing. WGS);
c) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific antigens);
d) morphological characteristics (e.g. round, long spindle);
e) histology (e.g. extracellular cellular markers).
Morphological characteristics and cell markers are useful to provide supporting data for occurrence of
cell cross-contamination, but are not suitable for authentication testing when used alone.
4.3.2.2 Sequence-specific STR or SNP profiles can be used to discriminate among individuals within the
same species. STR or SNP profiling-based Sanger sequencing or MPS technologies should be used for
detection of cell line identity but they also can provide data about intra-species cross-contamination.
Attention should be given to contamination at an early stage, which can go unnoticed even by these
techniques.
4.4 Identification of cell-line-specific characteristics
4.4.1 Detection of cell line genome heterogeneity
4.4.1.1 During extended in vitro cell culturing, cell lines can acquire additional genomic changes and
evolve into multiple genetically, transcriptionally, proteotypically or phenotypically different sub-clones
(e.g. the genetic instability and cell heterogeneity of cancer cell lines).
NOTE 1 Detection of cell line genome heterogeneity is not an authentication test method. Methods for cell line
authentication can be used for detecting cell line genome heterogeneity.
NOTE 2 It is reported that stocked HeLa cells originated from different laboratories show notable variability in
genome, steady-state mRNA expression, protein expression and protein turnover rates at uniform culture condition.
Moreover, progressive divergence can be observed within a specific HeLa cell line after three months of continuous
culture. The in vitro cell manipulating procedures, such as transfection and gene editing, can also lead to genetic
[2] [15]
heterogeneity . .
Detection of genomic changes depends on cell-line-specific markers, which can include:
a) genetic sequence (e.g. Sanger sequencing or MPS of DNA);
b) genetic characteristics (e.g. STR profiling, SNP profiling);
c) transcription (e.g. mRNA);
d) karyotype.
4.4.1.2 The detection methods of cell line gene mutations can include high-throughput sequencing (e.g.,
whole genome sequencing. WGS) along with karyotype analysis. The COSMIC database provides list of
[3] [16]
cell line somatic mutations found in various human cancers . .
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ISO/DTS 23511:2023(E)
4.4.2 Detection of cellular differentiation
4.4.2.1 Both pluripotent and multipotent stem cells can differentiate, either spontaneously or with
external stimuli into certain cell types in vitro. Cellular differentiation within a given cell line can be
detected by various gene expression markers, which can include, but are not limited to:
a) cell surface markers;
b) transcription factors;
c) signalling pathway-related intracellular markers;
d) enzymatic markers.
4.4.2.2 Flow cytometry analysis, immunofluorescent staining and enzyme-linked immunosorbent
assays can be used to detect differentiated cell–-specific gene expression, along with gene expression
assays, indicating cellular differentiation status.
NOTE Measurement of cellular differentiation can provide useful supporting data as part of broader cell line
identification, but is not suitable for authentication testing when used alone as this relies on genome-based
methods.
5 Application scenarios of cell line authentication
To avoid cell line misidentification and cross-contamination, cell line authentication should be used in
the following scenarios:
a) authentication and characterization of newly established cell lines;
b) routine inspection of cell lines in culture and in stock, especially for rapid growing cell types, cells in
extended culture, cells with unusual phenotype, and cells after a selection/sorting process;
c) validation of cell line origin upon receiving from other facilities, before sending the material to other
facilities and prior to banking;
d) validation of a cell line origin after the preparation of a cell bank (i.e. seed stock);
e) authentication of cell lines used in both basic and clinical research when abnormalities were found
in the cultured cells, or after multiple passaging.
6 Sample preparation
6.1 Samples utilized for cell line authentication can include whole cells, deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA). Identification errors can arise due to errors when labelling or handling samples,
or cross-contamination of cells or DNA. Care to reduce the possibility of error(s) shall always be taken
when handling or labelling cells or DNA.
6.2 Cells used for sampling should be fully mixed and representative of culturing status. The cells used
for karyotype analysis should be undergoing active proliferation so that cells in metaphase of mitosis are
present for analysis.
6.3 DNA should be of sufficient quality and quantity for downstream assays.
NOTE Requ
...
FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 23511
ISO/TC 276
Biotechnology — General
Secretariat: DIN
requirements and considerations for
Voting begins on:
2023-02-27 cell line authentication
Voting terminates on:
Biotechnologie — Exigences et considérations générales relatives à
2023-04-24
l'authentification de la lignée cellulaire
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BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO-
ISO/DTS 23511:2023(E)
LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN-
DARDS TO WHICH REFERENCE MAY BE MADE IN
NATIONAL REGULATIONS. © ISO 2023
---------------------- Page: 1 ----------------------
ISO/DTS 23511:2023(E)
FINAL
TECHNICAL ISO/DTS
DRAFT
SPECIFICATION 23511
ISO/TC 276
Biotechnology — General
Secretariat: DIN
requirements and considerations for
Voting begins on:
cell line authentication
Voting terminates on:
Biotechnologie — Exigences et considérations générales relatives à
l'authentification de la lignée cellulaire
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Email: copyright@iso.org
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO
ISO/DTS 23511:2023(E)
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LOGICAL, COMMERCIAL AND USER PURPOSES,
DRAFT INTERNATIONAL STANDARDS MAY ON
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OCCASION HAVE TO BE CONSIDERED IN THE
LIGHT OF THEIR POTENTIAL TO BECOME STAN
DARDS TO WHICH REFERENCE MAY BE MADE IN
ii
© ISO 2023 – All rights reserved
NATIONAL REGULATIONS. © ISO 2023
---------------------- Page: 2 ----------------------
ISO/DTS 23511:2023(E)
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Principles of cell line authentication .4
4.1 General . 4
4.2 Confirmation of cell origin . 4
4.3 Detection of crosscontamination . 4
4.3.1 Detection of cell line interspecies crosscontamination . 4
4.3.2 Detection of cell line intraspecies crosscontamination . 5
4.4 Identification of cell-line-specific characteristics. 5
4.4.1 Detection of cell line genome heterogeneity . 5
4.4.2 Detection of cellular differentiation . 6
5 Application scenarios of cell line authentication . 6
6 Sample preparation .7
7 Method options for cell line authentication . 7
7.1 General . 7
7.2 DNAbased cell line authentication methods . 7
7.2.1 Short tandem repeat profiling. 7
7.2.2 Single nucleotide polymorphism profiling . 9
7.2.3 DNA barcoding . 9
7.2.4 Multiplex PCR . 10
7.2.5 Whole genome sequencing . 10
7.3 Related methods for cell line identification . 10
8 Authentication method selection .11
8.1 General . 11
8.2 Cell origin . 11
8.2.1 Confirmation of cell line origin. 11
8.2.2 Identification of cell line gene mutations . 11
8.2.3 Identification of cell-line-specific properties . 11
8.3 Species types of cross-contamination . 11
8.3.1 Cell line interspecies crosscontamination . 11
8.3.2 Cell line intraspecies crosscontamination .12
8.4 Cell culture methods . 12
8.4.1 Authentication for cocultured cells .12
8.4.2 Authentication for ex vivo cell culture .12
8.4.3 Authentication for laboratory operation .12
8.5 Authentication purpose . 12
9 Quality control .13
9.1 Operator training . 13
9.2 Instruments and equipment .13
9.3 Reagents . 14
9.4 Validation and verification of methods . 14
9.4.1 General . 14
9.4.2 Validation . 14
9.4.3 Verification . 14
10 Report .15
10.1 Reporting . 15
10.2 Evaluation of measurement uncertainty . 15
iii
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ISO/DTS 23511:2023(E)
Annex A (informative) Detection methods for cell line authentication .16
Bibliography .18
iv
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ISO/DTS 23511:2023(E)
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and nongovernmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www.iso.org/directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www.iso.org/patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to
the World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see
www.iso.org/iso/foreword.html.
This document was prepared by Technical Committee ISO/TC 276, Biotechnology.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www.iso.org/members.html.
v
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ISO/DTS 23511:2023(E)
Introduction
Cell line authentication is a critical quality control (QC) procedure, which aims to verify a cell line’s
identity and show that it is free of contamination from other cell lines. It has been estimated that a
considerable proportion of the cell lines stored in the United States, Europe and Asia are misidentified
or cross-contaminated, which results in potentially misleading or non-repeatable data, causing
[13]
tremendous waste of time and effort. To facilitate proper utilization of a cell line, the standardization
of procedures used for cell line authentication is urgently needed. This document elaborates on general
requirements for cell line authentication based on the existing national standards and state-of-the-art
methods, aiming to represent and provide guidance to stakeholders in life science, biomedicine and
other related fields.
vi
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TECHNICAL SPECIFICATION ISO/DTS 23511:2023(E)
Biotechnology — General requirements and considerations
for cell line authentication
1 Scope
This document defines terms related to cell line authentication in the field of biotechnology. It describes
the general principles, detection strategies and analytical methods for cell line authentication. It
specifies requirements and key considerations for method selection, quality control parameters, data
analysis and reporting.
This document is applicable to routine inspection of cell lines in culture and in storage in the fields of
basic research, translational studies and product manufacturing. It is also applicable to cell line origin
validation in academic and industrial laboratories, cell banks and manufacturing sites. It is primarily
applicable for mammalian cells, including human cells.
This document does not apply to non-animal cells (e.g. microbial contamination, plant cells), nor to cells
in complex matrices (e.g. tissues, organs, organoids, plants).
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/IEC 17025, General requirements for the competence of testing and calibration laboratories
3 Terms and definitions
For the purposes of this document, the following terms and definitions apply.
ISO and IEC maintain terminology databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at https:// www .electropedia .org/
3.1
cell bank
collection of appropriate containers, whose contents are of uniform composition, stored under defined
conditions, and where each container represents an aliquot of a single pool of cells
[14]
[SOURCE: ICH Q5D ]
3.2
cell line
defined population of cells that has been passaged from a primary culture, and can be maintained in
culture for an extended period of time, retaining stability of certain initial phenotypes and functions
for its intended use
Note 1 to entry: A primary culture is a culture started from cells, tissues or organs taken directly from an
organism, and before the first subculture, propagation and consecutive passages in vitro.
1
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ISO/DTS 23511:2023(E)
3.3
cell line authentication
process by which the cell line (3.2) identity is verified and shown to be free of contamination from other
cell lines
3.4
cell line identification
process by which the cell line (3.2) identity is verified, which includes confirmation of cell origin, and
identification of cell-specific characteristics
3.5
microbial contamination
presence of exogenous:
a) bacteria and/or fungi;
b) viruses; and/or
c) foreign inter or intraspecies cell lines (3.2) in a cell culture
Note 1 to entry: Some cell lines have endogenous virus/viral sequences.
Note 2 to entry: Point c) is commonly known as “cell cross-contamination”.
3.6
detection limit
lowest quantity of a substance that can be distinguished from the absence of that substance with a
stated confidence limit
[SOURCE: ISO 14687:2019, 3.5]
3.7
DNA barcoding
taxonomic method that uses a short genetic marker in an organism’s DNA to identify it as belonging to
a particular species
3.8
immunofluorescence
method for studying the distribution of specific protein antigens in cells by combining immunological
methods (antigen-specific binding) with fluorescent labelling techniques
3.9
cell line inter-species cross-contamination
contamination of a cell culture by cells derived from different species
3.10
cell line intra-species cross-contamination
contamination of a cell culture by the same type of cells (from different individuals) or different types
of cells (from the same or different individuals) derived from the same species
3.11
isozyme analysis
isoenzyme analysis
separation technique based on electrophoresis to generate patterns of enzymatically active
polypeptides with identical specificity (3.19) but of different molecular structure
3.12
karyotype analysis
chromosomal analysis in each cell to detect aneuploidy, structural abnormalities and ploidy
2
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ISO/DTS 23511:2023(E)
3.13
MPS
massively parallel sequencing
sequencing technique based on the determination of incremental template based polymerization of
many independent DNA molecules simultaneously
Note 1 to entry: Massively parallel sequencing technology can provide millions or billions of short reads per run.
[SOURCE: ISO 203972:2021, 3.30]
3.14
cell line misidentification
incidence where the cell line (3.2) identity is incorrectly is given through mislabelling
3.15
PCR
polymerase chain reaction
enzymatic procedure which allows in vitro amplification of DNA
[SOURCE: ISO 22174:2005, 3.4.1]
3.16
sensitivity
quotient of the change in an indication of a measuring system and the corresponding change in a value
of a quantity being changed
[SOURCE: ISO/IEC Guide 99:2007, 4.12, modified ― Preferred term “sensitivity of a measuring system”
and notes to entry deleted. “changed” replaced “measured”.]
3.17
STR
short tandem repeat
variable segments of DNA that are composed of multiple adjacent two to five basepair long sequences
3.18
SNP
single nucleotide polymorphism
single nucleotide variation in a genetic sequence that occurs at an appreciable frequency in the
population
[SOURCE: ISO 25720:2009, 4.23]
3.19
specificity
property of a method to respond exclusively to the characteristic or analyte under investigation
[SOURCE: ISO 24276:2006, 3.1.4]
3.20
validation
confirmation, through the provision of objective evidence, that the requirements for a specific intended
use or application have been fulfilled
[SOURCE: ISO 9000:2015, 3.8.13, modified ― Notes to entry deleted.]
3.21
WGS
whole genome sequencing
methods that approach determination of the entire nucleotide sequence of the nuclear DNA of eukaryotic
organisms
3
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ISO/DTS 23511:2023(E)
3.22
verification
confirmation, through the provision of objective evidence, that specified requirements have been
fulfilled
[SOURCE: ISO 9000:2015, 3.8.12, modified ― Notes to entry deleted.]
4 Principles of cell line authentication
4.1 General
Multiple test methods that rely on genomic analysis combined with phenotypic characteristics can be
used as part of the process of cell line authentication. Purposes of genomic analysis include:
a) confirmation of cell origin;
b) examination of cell species, to ensure that no cell line inter- or intra-species cross-contamination
exists in cell cultures;
c) identification and/or confirmation of certain cell-line-specific characteristics. Cell-line-specific
characteristics such as gene mutations can be useful supporting evidence for the cell line
authentication. However, many “cell-specific” characteristics are related to tissue type or disease
status and are not unique.
4.2 Confirmation of cell origin
For a newly established cell line, a liquid or solid tissue sample from which a cell line is derived, or
a liquid or solid tissue sample from the same donor from whom the cell line was derived, should be
stored for origin confirmation. The baseline DNA profile of the original sample should be used in cell
line authentication by comparing it to the DNA profiles of subsequent passages. If the source tissue,
blood, or both are not available, the DNA profile of an early passage stock can be used as the baseline.
DNA-based profiling methods intended for routine genotype analysis include:
a) short tandem repeat (STR) analysis with polymerase chain reaction (PCR) assays followed by
fragment size analysis or by Sanger sequencing;
b) single nucleotide polymorphism (SNP) analysis by single-base extension assay or SNP genotyping
qPCR assays;
c) latest DNA profiling technologies, such as massively parallel sequencing (MPS).
SNP databases of targeted panels are now available for analysis. However, there are no central databases
or universally accepted SNP markers, so any SNP comparison shall be in-house or have similar usage
limitations. The whole genome sequencing (WGS) data of newly or already established cell lines should
be provided as a further information source.
4.3 Detection of cross-contamination
4.3.1 Detection of cell line inter-species cross-contamination
4.3.1.1 Cell line inter-species cross-contamination occurs when a cell line is contaminated by
undesired cells from different species. Cell lines derived from different species have different
characteristics, not all of which are suitable for authentication.
Cell line authentication should be performed with consideration for various characteristics, including:
a) genetic characteristics (e.g. COI, CytB and ND5 genes);
b) cytogenetic characteristics (e.g. chromosome karyotype, marker chromosome);
4
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ISO/DTS 23511:2023(E)
c) biochemical characteristics (e.g. enzyme type);
d) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific
antigens);
e) cell kinetics (e.g. differences in cell division frequency or cell generation time);
f) morphological characteristics (e.g. round, long spindle).
4.3.1.2 Methods based on different measurement principles should be used for the detection of cell
line inter-species cross-contamination. For genetic and cytogenetic characteristics, detection methods
include DNA barcoding, PCR assays and karyotype analysis. DNA barcoding can be used to investigate
the mitochondrial gene sequences associated with species-specific cytochrome c oxidase subunit 1
(CO1) gene. PCR assays utilize either species-specific or degenerate primers, which can amplify DNA
fragments for species identification and can detect lower levels of cross-contamination than Sanger
sequencing-based DNA barcoding. Karyotype analysis can directly reveal cross-contamination by
comparing species-specific chromosomes. Morphological characteristics, cell kinetics, biochemical
characteristics and phenotype are useful to provide supporting data for occurrence of cell cross-
contamination, but are not suitable for authentication testing when used alone.
4.3.2 Detection of cell line intra-species cross-contamination
4.3.2.1 Cell line intra-species cross-contamination occurs when a cell line is contaminated by cells of
the same type (from different individuals) or of different types (from the same or different individuals)
within the same species. Detection of cell line intraspecies crosscontamination depends on individual
cell-line-specific characteristics, which can include:
a) genetic characteristics (e.g. STR profiling, SNP profiling);
b) genetic sequence (e.g. WGS);
c) cell markers (e.g. proteins, lipids, glycosylation, histocompatibility antigen, tissue-specific
antigens);
d) morphological characteristics (e.g. round, long spindle);
e) histology (e.g. extracellular cellular markers).
Morphological characteristics and cell markers are useful to provide supporting data for occurrence of
cell crosscontamination, but are not suitable for authentication testing when used alone.
4.3.2.2 Sequence-specific STR or SNP profiles can be used to discriminate among individuals within
the same species. STR or SNP profiling-based Sanger sequencing or MPS technologies should be used for
detection of cell line identity but they also can provide data about intra-species cross-contamination.
Attention should be given to contamination at an early stage, which can go unnoticed even by these
techniques.
4.4 Identification of cell-line-specific characteristics
4.4.1 Detection of cell line genome heterogeneity
4.4.1.1 During extended in vitro cell culturing, cell lines can acquire additional genomic changes
and evolve into multiple genetically, transcriptionally, proteotypically or phenotypically different sub-
clones (e.g. the genetic instability and cell heterogeneity of cancer cell lines).
NOTE 1 Detection of cell line genome heterogeneity is not an authentication test method. Methods for cell line
authentication can be used for detecting cell line genome heterogeneity.
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ISO/DTS 23511:2023(E)
NOTE 2 It is reported that stocked HeLa cells originated from different laboratories show notable variability
in genome, steady-state mRNA expression, protein expression and protein turnover rates at uniform culture
condition. Moreover, progressive divergence can be observed within a specific HeLa cell line after three months
of continuous culture. The in vitro cell manipulating procedures, such as transfection and gene editing, can also
[15]
lead to genetic heterogeneity.
Detection of genomic changes depends on cell-line-specific markers, which can include:
a) genetic sequence (e.g. Sanger sequencing or MPS of DNA);
b) genetic characteristics (e.g. STR profiling, SNP profiling);
c) transcription (e.g. mRNA);
d) karyotype.
4.4.1.2 The detection methods of cell line gene mutations can include high-throughput sequencing
(e.g. WGS) along with karyotype analysis. The COSMIC database provides list of cell line somatic
[16]
mutations found in various human cancers.
4.4.2 Detection of cellular differentiation
4.4.2.1 Both pluripotent and multipotent stem cells can differentiate, either spontaneously or with
external stimuli into certain cell types in vitro. Cellular differentiation within a given cell line can be
detected by various gene expression markers, which can include, but are not limited to:
a) cell surface markers;
b) transcription factors;
c) signalling pathway-related intracellular markers;
d) enzymatic markers.
4.4.2.2 Flow cytometry analysis, immunofluorescent staining and enzyme-linked immunosorbent
assays can be used to detect differentiated cell-specific gene expression, along with gene expression
assays, indicating cellular differentiation status.
NOTE Measurement of cellular differentiation can prov
...
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