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

General Information

Status

Published

Publication Date

17-May-2023

ICS

07.080 - Biology. Botany. Zoology

Technical Committee

ISO/TC 276 - Biotechnology

Drafting Committee

ISO/TC 276/WG 3 - Analytical methods

Current Stage

6060 - International Standard published

Start Date

18-May-2023

Due Date

02-Jul-2023

Completion Date

18-May-2023

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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

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
© ISO 2023 – All rights reserved
---------------------- Page: 2 ----------------------
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
© ISO 2023 – All rights reserved
---------------------- Page: 3 ----------------------
ISO/TS 23511:2023(E)

Annex A (informative) Detection methods for cell line authentication .....................................................................16

Bibliography .............................................................................................................................................................................................................................18

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
---------------------- Page: 5 ----------------------
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.
© ISO 2023 – All rights reserved
---------------------- Page: 6 ----------------------
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.

© ISO 2023 – All rights reserved
---------------------- Page: 7 ----------------------
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

© ISO 2023 – All rights reserved
---------------------- Page: 8 ----------------------
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
© ISO 2023 – All rights reserved
---------------------- Page: 9 ----------------------
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);
© ISO 2023 – All rights reserved
---------------------- Page: 10 ----------------------
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.
© ISO 2023 – All rights reserved
---------------------- Page: 11 ----------------------
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.
© ISO 2023 – All rights reserved
---------------------- Page: 12 ----------------------
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

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

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
---------------------- Page: 4 ----------------------
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
---------------------- Page: 5 ----------------------
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
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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

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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)

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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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
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l'authentification de la lignée cellulaire
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ISO/DTS 23511:2023(E)
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SPECIFICATION 23511
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Biotechnology — General
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requirements and considerations for
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cell line authentication
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Contents Page

4 Principles of cell line authentication .............................................................................................................................................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

7 Method options for cell line authentication ............................................................................................................................ 7

7.2 DNAbased cell line authentication methods .............................................................................................................. 7