ISO/TS 23511:2023

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 2023
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ii
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
Annex A (informative) Detection methods for cell line authentication .16
Bibliography .18
iv
Foreword
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v
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
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.
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
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
3.22
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);
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.
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 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 Requirements can vary with the authentication methods.
6.4 For DNA extraction kits, the supplier’s technical manual or other instructions shall be followed. A
validation is recommended in order to define the ideal quantity of DNA to be used in the test so that the
true peaks are well defined. ISO 20395 can be used for information on more detailed procedures.
7 Method options for cell line authentication
7.1 General
Each method for cell line authentication has its intrinsic deficiencies, which can affect applicability
and accuracy (as described in Annex A). Users can select one or more methods depending on their
information of cell type, application scenario, sample preparation or potential contaminating sources.
NOTE 1 Historical methods such as human leukocyte antigen (HLA) profiling by PCR and isozyme analysis
have previously been used for cell line authentication, but are no longer used due to their limitations in
applicability, sensitivity and accuracy. Currently, DNA-based methods are most widely used for cell line
authentication, which include STR profiling, SNP profiling, HLA sequencing, DNA barcoding and
...