ISO 24603:2022

Biotechnology - Biobanking - Requirements for human and mouse pluripotent stem cells

This document specifies requirements for the biobanking of human and mouse pluripotent stem cells (PSCs), including the collection of biological source material and associated data, establishment, expansion, characterization, quality control (QC), maintenance, preservation, storage, thawing, disposal, distribution and transport. This document is applicable to all organizations performing biobanking with human and mouse PSCs used for research and development. This document does not apply to cell lines used for in vivo application in humans, clinical applications or therapeutic use. NOTE International, national or regional regulations or requirements, or multiple of them, can also apply to specific topics covered in this document.

Biotechnologie — Biobanking — Exigences relatives aux cellules souches pluripotentes humaines et murines

General Information

• Status: Published
• Publication Date: 02-Aug-2022
• ICS: 07.080 - Biology. Botany. Zoology
• Technical Committee: ISO/TC 276 - Biotechnology
• Drafting Committee: ISO/TC 276/WG 2 - Biobanks and bioresources
• Current Stage: 6060 - International Standard published
• Start Date: 03-Aug-2022
• Due Date: 07-Oct-2022
• Completion Date: 03-Aug-2022

Overview

ISO 24603:2022 - "Biotechnology - Biobanking - Requirements for human and mouse pluripotent stem cells" specifies quality and operational requirements for biobanking of human and mouse pluripotent stem cells (PSCs). It covers the full biobank lifecycle: collection of biological source material and associated data, establishment and expansion of cell lines, characterization and quality control (QC), preservation (cryopreservation), storage, thawing, disposal, distribution and transport. The standard is intended for organizations performing PSC biobanking for research and development and explicitly excludes cell lines intended for in vivo human clinical or therapeutic use.

Key topics and technical requirements

ISO 24603 organizes practical requirements around core biobanking activities and governance, including:

• Collection and donor information: requirements for associated donor data (human and mouse), ethical and legal considerations, and informed traceability.
• Facility, personnel and supplies: competency of staff, appropriate facilities and validated reagents/consumables.
• Establishment and expansion: procedures for generating PSC lines, unique identification and cell line master files.
• Characterization and functionality: testing for pluripotency, differentiation potential, population doubling time, genetic integrity and culture stability.
• Quality control and testing: microbial contamination checks, testing for infectious agents and routine QC procedures.
• Preservation and storage: cryopreservation best-practice, storage conditions and inventory management to ensure viability and provenance.
• Thawing, distribution and transport: validated thawing procedures and controlled transport/distribution with required data for end-users.
• Documentation and data management: robust information management, traceability, and record keeping (e.g., cell line master file).
• Annexes: informative guidance on potency comparison, example culture/establishment methods and a QC procedure template.

Applications and who uses this standard

ISO 24603 is used by:

• Biobanks and stem cell resource centers that store and manage human PSCs and mouse PSCs.
• Academic and industry research laboratories generating or sourcing PSC lines for basic research, disease modelling, organoid development and drug screening.
• Quality assurance, laboratory managers and regulatory affairs teams seeking harmonized biobanking standards and reproducible cell line data.
• Suppliers of biobanking services, sample transporters and custodians requiring traceability and validated handling practices.

This standard helps organizations improve reproducibility, protect sample integrity, and manage ethical and legal obligations while facilitating safe exchange of PSC materials for R&D.

Related standards

• ISO 20387:2018 - General requirements for biobanking
• ISO/TS 20388:2021 - Requirements for animal biological material
• ISO 21709:2020 - Process and quality requirements for mammalian cell lines
• ISO 8601-1 (date/time representation) and other ISO/TS documents referenced in ISO 24603

Keywords: ISO 24603, biobanking, pluripotent stem cells, human PSCs, mouse PSCs, quality control, cell line characterization, cryopreservation, stem cell biobank requirements.

Standards Content (Sample)

INTERNATIONAL ISO
STANDARD 24603
First edition
2022-08
Biotechnology — Biobanking —
Requirements for human and mouse
pluripotent stem cells
Biotechnologie — Biobanking — Exigences relatives aux cellules
souches pluripotentes humaines et murines
Reference number
© ISO 2022
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
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or ISO’s member body in the country of the requester.
ISO copyright office
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Phone: [phone]
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Published in Switzerland
ii
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 5
5 General requirements . 6
5.1 General . 6
5.2 Legal and ethical requirements . 6
5.3 Personnel, facilities and equipment . 6
5.4 Reagents, consumables and other supplies . 7
5.5 Management of information and data . 7
6 Collection of biological source materials and associated data. 8
6.1 Information about the human donor and requirements for the biological material . 8
6.2 Information about the mouse donor and requirements for the biological material . 8
6.3 Collection procedure . 9
7 Transport of the biological source material or PSCs and associated data to the
biobank . 9
8 Reception and traceability of the biological source material or PSCs and associated
data .10
9 Establishment of cell lines .10
9.1 Processes . 10
9.2 Unique identification . 10
9.3 Testing for infectious agents . 10
10 Characterization .11
10.1 General . 11
10.2 Population doubling time and subculture/passage.12
10.2.1 PDT . 12
10.2.2 Subculture/passage .13
10.3 Stability of the culture .13
10.4 Functionality . 14
10.4.1 General . 14
10.4.2 In vitro differentiation . 14
10.5 Microbial contamination . 14
11 Quality control .14
12 Testing.15
13 Cell line management .15
14 Preservation of cell lines .15
15 Storage .16
16 Thawing .16
17 Disposal .17
18 Distribution .17
18.1 General requirements . 17
18.2 Information for users . 17
18.3 Transport . 18
Annex A (informative) Potency comparison .21
iii
Annex B (informative) Examples for methods for the establishment and culture of PSCs .22
Annex C (informative) Quality control procedure for biobanking of human and mouse PSCs .26
Bibliography .27
iv
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
Introduction
Pluripotent stem cells (PSCs), including embryonic stem cells (ESCs) and induced pluripotent stem cells
(iPSCs), have been extensively studied in scientific research in order to improve the understanding
of developmental biology and diseases, to create organoids for drug screening, and to be applied in
cell-based therapies. In just a few years, thousands of PSC lines have been established in laboratories
around the world. PSC lines hold unique characteristics and behaviour due to their capability for both
self-renewal and differentiation into multiple cell types. However, the stem cell phenotype can be
changed by suboptimal cell culture technique, prolonged passage or changing the culture conditions.
Clearly the consequences of using adversely affected cells would be wasted time and resources but,
even more seriously, the generation of erroneous data in the literature which could both confuse
and delay scientific progress in this area. Accordingly, mouse PSCs have been used to establish our
fundamental understanding of PSC biology and these discoveries have been translated into human PSC
research to drive the development of new human-cell-based in vitro assays and potential regenerative
medicines. Mouse PSCs and human PSCs have become the most widely studied species in this field and
many significant scientific advances have been made by using PSCs from these two species. Of course,
PSC lines have been established from other species such as rat, porcine, canine, bovine, primate, etc.
and those from primates in particular have provided understanding of the biology of these cells which
can be more relevant to human stem cell biology than data from mouse PSCs. However, PSCs from
these species are much less used in research laboratories than mouse and human and are therefore not
described specifically in this document although much of this document will be relevant to them.
Human PSCs developed in research environments will give the clues to the development of cell therapies,
thus ensuring that cell lines used in this dynamic field have been prepared and documented appropriately
and have the correct identity and characteristics, which is critical to help ensure reproducibility in PSC-
based research. This document aims to meet the current demand for standardized PSC procedures
[9]
of biobanks and builds on international consensus agreed by PSC resource centres . This document
specifies the establishment, maintenance, characterization, storage and distribution requirements for
mouse and human PSCs, providing a general guideline for both biobanking and fundamental research
of PSCs.
vi
INTERNATIONAL STANDARD ISO 24603:2022(E)
Biotechnology — Biobanking — Requirements for human
and mouse pluripotent stem cells
1 Scope
This document specifies requirements for the biobanking of human and mouse pluripotent stem
cells (PSCs), including the collection of biological source material and associated data, establishment,
expansion, characterization, quality control (QC), maintenance, preservation, storage, thawing,
disposal, distribution and transport.
This document is applicable to all organizations performing biobanking with human and mouse PSCs
used for research and development.
This document does not apply to cell lines used for in vivo application in humans, clinical applications or
therapeutic use.
NOTE International, national or regional regulations or requirements, or multiple of them, can also apply to
specific topics covered in this document.
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 8601-1, Date and time — Representations for information interchange — Part 1: Basic rules
ISO 20387:2018, Biotechnology — Biobanking — General requirements for biobanking
ISO/TS 20388:2021, Biotechnology — Biobanking — Requirements for animal biological material
ISO 21709:2020, Biotechnology — Biobanking — Process and quality requirements for establishment,
maintenance and characterization of mammalian cell lines
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 20387:2018 and the following
apply.
ISO and IEC maintain terminological 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
biobank
legal entity or part of a legal entity that performs biobanking (3.2)
[SOURCE: ISO 20387:2018, 3.5]
3.2
biobanking
process of acquisitioning and storing, together with some or all of the activities related to collection,
preparation, preservation, testing, analysing and distributing defined biological material as well as
related information and data
[SOURCE: ISO 20387:2018, 3.6]
3.3
cell line master file
complete dossier of all procedures and records used to generate and maintain a cell line
3.4
cell morphology
form and structure of the cell
Note 1 to entry: Morphology can be represented by a single parameter or a combination of two or more
parameters.
[SOURCE: ISO 21709:2020, 3.3]
3.5
cell population purity
percentage of a particular cell type in a population, of which has the same specific biological
characteristics, such as cell surface markers, genetic polymorphisms and biological activities
[SOURCE: ISO/TS 22859:2022, 3.8]
3.6
cryopreservation
process by which cells are maintained in an ultra-low temperature in an inactive state so that they can
be revived later
[SOURCE: ISO 21709:2020/Amd 1:2021, 3.6]
3.7
differentiation
process to bring the cells into a defined cell state or fate
[SOURCE: ISO/TS 22859:2022, 3.11]
3.8
differentiation potential
ability that refers to the concept that stem and progenitor cells can produce daughter cells which are
able to further differentiate into other cell types
[SOURCE: ISO/TS 22859:2022, 3.12]
3.9
embryonic stem cell
ESC
pluripotent stem cell (3.21) derived from the inner cell mass of a blastocyst, i.e. an early stage pre-
implantation embryo
3.10
ethics review committee
body which is responsible for the evaluation and review of the ethical issues involved in the research
3.11
expansion
cell culturing process by which the cell number increases in vitro
3.12
feeder cell
mitotically inactivated cell used to support the growth of pluripotent stem cells (3.21)
3.13
genetic integrity
genome of cells that has not been altered
3.14
genetic state
phenotype of genetic profile of individual organism, including but not limited to karyotype (3.18),
integrity, mutation and knock-in of exogenous sequence
3.15
harvest
process of obtaining cells from a cell culture environment
3.16
identity verification
part of the process of verifying authenticity of a cell line in which cell origin is genetically confirmed
[SOURCE: ISO 21709:2020, 3.10]
3.17
induced pluripotent stem cell
iPSC
pluripotent stem cell (3.21) that is generated from somatic cells through artificial reprogramming by
the introduction of genes or proteins, or via chemical or drug treatment
3.18
karyotype
characteristics of the chromosomes of a cell, including its number, type, shape and structure, etc.
3.19
passage
subculture
process of further culturing of cells in a culture vessel to provide higher surface area/volume for the
cells to grow
Note 1 to entry: A passage can be performed by harvesting an aliquot from the parent vessel and reseeding it into
another vessel.
[SOURCE: ISO/TS 22859:2022, 3.18]
3.20
passage number
number of subculturing that occurred
Note 1 to entry: For this document, P is understood as the starting population of the cells.
[SOURCE: ISO 21709:2020, 3.13, modified — Note 1 to entry added.]
3.21
pluripotent stem cell
PSC
stem cell (3.26) that can differentiate into all cell types of the body and is able to self-renew indefinitely
in vitro
Note 1 to entry: PSCs include embryonic stem cells (ESCs) (3.9) (including fertilization derived ESCs, somatic cell
nuclear-transferred stem cells (3.25), etc.) and induced pluripotent stem cell (iPSCs) (3.17).
Note 2 to entry: ESC-like cells can also be isolated by parthenogenetic division of oocytes or other haploid cell
sources, and these cells have many of the characteristics of ESCs. However, certain features of these pluripotent
cell types can require specific characterization approaches.
3.22
population doubling time
PDT
doubling time
time taken for cultured cell count to double
Note 1 to entry: The time is measured in hours.
[SOURCE: ISO 21709:2020, 3.8, modified — “population doubling time” and “PDT” added as the preferred
term. Note 1 to entry added.]
3.23
self-renewal
ability of stem cells (3.26) to divide symmetrically, forming two identical daughter stem cells
Note 1 to entry: Adult stem cells can also divide asymmetrically to form one daughter cell which can proceed
irreversibly to a differentiated cell lineage and ultimately lead to specialized functional differentiated cells, while
the other daughter cell still retains the characteristics of the parental stem cell.
[SOURCE: ISO/TS 22859:2022, 3.23]
3.24
separation
process of obtaining target cells from biological samples
3.25
somatic cell nuclear-transferred stem cell
embryonic stem cells (3.9) derived from in vitro transfer of a donor cell nucleus into an enucleated oocyte
3.26
stem cell
non-specialized cells with the capacity for self-renewal (3.23) and differentiation potential (3.8), which
can differentiate into one or more different types of specialized cells
Note 1 to entry: Based on potency, stem cells can be divided into: totipotent stem cell (3.29), pluripotent stem cell
(3.21), multipotent stem cell, oligopotent stem cells, and unipotent stem cells (see Annex A).
[SOURCE: ISO/TS 22859:2022, 3.24, modified — Note 1 to entry replaced.]
3.27
stem cell marker
protein or gene specifically expressed in stem cells (3.26), usually used to isolate and identify stem cells
Note 1 to entry: Stem cell markers vary depending on stem cell type.
3.28
teratoma
tumour containing representative differentiated tissues and cells from the three germ layers
3.29
totipotent stem cell
stem cell (3.26) that can differentiate into an intact new organism including embryonal and extra
embryonal cells
3.30
viability
attribute of being alive (e.g., metabolically active, capable of reproducing, have intact cell membrane, or
have the capacity to resume these functions)
[SOURCE: ISO 21709:2020, 3.17, modified — “as defined based on the intended use” deleted.]
4 Abbreviated terms
bFGF basic fibroblast growth factor
EMRO embryo research oversight
ESC embryonic stem cell
HBV hepatitis B virus
HCMV human cytomegalovirus
HCV hepatitis C virus
HIV human immunodeficiency virus
HLA human leukocyte antigen
HTLV human T-lymphotropic virus
IFU instructions for use
iPSC induced pluripotent stem cell
KLF4 krueppel-like factor 4
KSR knockout serum replacement
mLIF mouse leukaemia inhibitory factor
MTA materials transfer agreement
OCT4 octamer-binding transcription factor 4
OriP origin of replication
PBMC peripheral blood mononuclear cell
PSC pluripotent stem cell
QC quality control
SOX2 SRY (sex determining region Y)-box 2
SSEA3 stage-specific embryonic antigen 3
SSEA4 stage-specific embryonic antigen 4
SSEA1 stage-specific embryonic antigen 1
STR short tandem repeat
SV40LT Simian virus 40 large T
TP treponema pallidum
5 General requirements
5.1 General
The biobank shall follow ISO 20387 and ISO 21709, in addition to this document. ISO/TR 22758 can be
used as additional reference for the implementation of ISO 20387. For mouse PSCs, ISO/TS 20388 shall
also be followed.
The biobank shall establish criteria and procedures for the isolation, establishment, expansion, storage,
thawing and transport of PSCs.
A data analysis procedure shall be established, documented, implemented, regularly reviewed and
updated.
The biobank shall use validated and/or verified methods and procedures for activities pertaining to
PSCs in accordance with ISO 20387:2018, 7.9.2 and 7.9.3, at all stages of the biological material life cycle
(as defined in ISO 20387:2018, 3.29).
According to the characteristics of PSCs, procedures, QC documents for collection, separation,
expansion, storage, transportation and testing, and data analysis shall be established, documented,
implemented, regularly reviewed and updated.
5.2 Legal and ethical requirements
ISO 20387:2018, 4.1.6, 4.3, 7.2.3.4, 7.3.2.4, A.7 a), and ISO 21709:2020, 4.2, shall be followed. For mouse
PSCs, ISO/TS 20388:2021, 4.2, shall also be followed.
The biobank shall collect relevant information on ethical requirements, implement and ‎regularly
update them, where relevant.
It is important to recognize that PSC lines are potentially not acceptable for use in research or
development or both in some countries, and shipment of cells to collaborating organizations will require
consideration of these differences. The biobank shall establish, document and implement policies on the
procurement and supply of PSCs.
Experimental plans using or establishing human PSCs should be consulted in a specialized ethics
review committee with particular expertise in topics relevant to the type and intended use of the PSC
lines in the biobank.
The biobank shall establish a process to verify and document cell line provenance, to be able to provide
evidence of ethical and regulatory compliance.
The biobank shall be aware whether reimbursement was made for the donation of human embryos/
tissues and whether the human embryo was created for research as this can be illegal in some countries.
For derivation of new pluripotent cell lines from human embryos, the ethical review process shall refer
to relevant expert ethical reviews.
[10]
EXAMPLE The human embryo research oversight (EMRO) process (ISSCR guidelines 2016, Chapter 2.1) .
Ethical requirements relevant for distribution are provided in 18.1.
5.3 Personnel, facilities and equipment
ISO 20387:2018, Clause 6, and ISO 21709:2020, 4.3, 4.4, 4.7, shall be followed. For mouse PSCs,
ISO/TS 20388:2021, 4.3, shall also be followed.
The biobank personnel shall be appropriately and specifically trained in PSC generation,
characterization, culture, cryopreservation, thawing and transport.
The biobank shall ensure that external operators providing PSC services demonstrate relevant
knowledge, experience and corresponding skills.
The biobank shall ensure that facilities, equipment and environmental conditions do not adversely
affect PSC quality attributes or invalidate the test results.
Equipment management procedures should be established, including the use of equipment and
maintenance plan.
The biobank shall control the operating environment and conditions (e.g. temperature, humidity,
cleanliness) according to the relevant characteristics of PSCs and the need for aseptic processing.
5.4 Reagents, consumables and other supplies
ISO 21709:2020, 4.5, shall be followed. For mouse PSCs, ISO/TS 20388:2021, 5.1.3, shall also be followed.
The biobank shall establish acceptance criteria for materials, including reagents and consumables,
necessary for PSC isolation, establishment, expansion, preservation, storage, thawing and transport.
If animal serum is used for PSC culture, there should be no evident potential high risk source of virus
or bovine spongiform encephalopathy, which cannot be managed by a risk assessment of the biological
source material and decontamination (such as irradiation for certain viruses).
For culture of human cell lines, if there are blood components in the culture medium (such as platelet
lysate, serum, albumin, transferrin and various cytokines), the source, batch number and quality
verification report shall be documented; and, if possible, a risk assessment shall be completed following
communication with the manufacturer on the risk of microbial contamination and other potential
hazards such as toxic contaminants. Where approved sources of these components are available, they
shall be used unless unsuitable for technical or logistical reasons.
5.5 Management of information and data
ISO 20387:2018, 7.8.3 and 7.10, shall be followed.
The biobank shall manage and maintain associated data of PSC lines, including but not limited to the
following:
a) the technical information: methods used in the derivation of cells/lines, culture conditions, passage
data including the passage number, characterization and microbiological test data;
b) the preservation and storage information;
c) the safety testing data;
d) the cell identity verification methods, e.g. by short tandem repeat (STR) analysis and/or HLA-
typing or equivalent validated methods.
Certain data retention times, data integrity and security of data storage shall be ensured.
For human PSCs, a minimum period of retention of records shall be established. Special requirements
for storage and retention times can apply for future applications. Personal data of each human donor
shall be held in a protected location and shall be handled in accordance with ISO 20387:2018, 4.3.
The cell line master file shall be kept to enable review of the data and records for specific applications.
6 Collection of biological source materials and associated data
6.1 Information about the human donor and requirements for the biological material
A risk assessment shall be performed and documented.
To protect the private data of the donor, the biobank shall establish donor data protection methods in
accordance with ISO 20387:2018, 4.3.
The documentation of the donor information shall be performed. Where possible, the documentation
shall be performed prior to sample collection. The documentation shall include but is not limited to:
a) the donor reference, which can be in form of a code (e.g. pseudonymized, anonymized);
b) the relevant health status of the donor (e.g. medical history, statement of donor health or suitability,
disease type, concomitant disease, demographics such as age and sex);
NOTE The ABO blood groups and category classification data of HLA of the donor can also be collected
depending on the situation.
c) the information about medical treatment and special treatment prior to the collection (e.g. date,
terms of treatment, medication, conclusion of medical specialist);
d) where applicable, information about the informed consent given by the donor (e.g. copy of the signed
informed consent signature form with details of the donors’ name redacted); see ISO 20387:2018,
7.2.3.4.
Documentation of the donor information should include the geographical region of the donor as needed
based on the purpose of research.
For planned iPSC line establishment, the documentation of donor information shall include but is not
limited to:
— sex;
— age;
— tissue or cell type.
During the collection process for human cells, measures shall be taken to protect donor and biobank
personnel health and safety.
6.2 Information about the mouse donor and requirements for the biological material
The biobank shall establish, document and implement inclusion and exclusion criteria based on the
purpose of research.
Documentation of the mouse donor information shall take place prior to collection, shall include a) and
b), and should include, but is not limited to c) and d) of the following list:
a) the strain and genotype;
b) the demographics (i.e. age and sex, etc.);
c) the relevant health status of the mouse (e.g. statement of donor health or suitability, disease type,
concomitant disease);
d) the information about medical treatment and special treatment prior to the collection (e.g. date,
terms of treatment, medication, conclusion of medical specialist, stress, diet).
The ID of the mouse, which can be in form of a code (e.g. according to Reference [11]), should be
additionally documented, if available. For planned iPSC line establishment, the documentation of donor
information shall additionally include but is not limited to:
— sex;
— age;
— tissue or cell types;
— the reprogramming strategies including reprogramming factors and gene delivery system.
The animal welfare requirements of donor mouse husbandry should conform to ISO 10993-2:2006.
If the mouse PSC is established from a specific strain or genetic modified mouse developed by another
laboratory, company or organization, an agreement for new established mouse PSCs shall be required
with the mouse donor’s legally designated representative.
If the donated mouse is genetically modified, its use does not necessarily conflict with the original
owner’s rights.
For PSCs from mice, any known colony infectious agent screening shall be documented.
6.3 Collection procedure
ISO 20387:2018, 7.2, shall be followed.
The biobank shall establish, implement, validate and document a collection procedure for each relevant
biological source material.
NOTE Each selected tissue has specific requirements for collection and best practice. Taking into account
new developments can improve the quality of harvested cells.
All reagents and materials used to collect the biological material shall be sterile.
[12]
The biobank should conform to ISO 35001 or the WHO’s Laboratory Biosafety Manual when handling
biological material contaminated with pathogens.
The risk of microbiological contamination (bacterial, fungal, viral, parasitic) should be mitigated by
focusing on those agents which are most likely to be contaminants in relation to the geography, donor
cohort and tissue being procured.
7 Transport of the biological source material or PSCs and associated data to the
biobank
ISO 20387:2018, 7.4, shall be followed. ISO/TS 20658 can be used to consider transport and handling,
and safety requirements for facilities. For mouse PSCs, ISO/TS 20388:2021, Clause 6, shall also be
followed.
[12]
The biobank should conform to ISO 35001 or the WHO’s Laboratory Biosafety Manual when handling
biological material contaminated with pathogens.
The biobank shall determine the appropriate conditions for the transportation of the biological source
material from the collection facility to the biobank. Instructions on the transportation of biological
source material to the preparation site as well as the transportation of PSC preparations to the biobank
should be included.
The following factors shall be taken into account for transportation:
a) packaging, material, containers and secondary containment;
b) medium or solvent;
c) transportation duration and temperature.
Biological source material storage media and conditions shall be established, implemented, documented
and validated to ensure maintenance of the viability and other key parameters.
The sample shall be transported under appropriate biosafety conditions.
A procedure for critical control points shall be established, implemented and documented.
8 Reception and traceability of the biological source material or PSCs and
associated data
ISO 20387:2018, 7.3.1, 7.3.2 and 7.5, shall be followed. For mouse PSCs, ISO/TS 20388:2021, Clause 7,
shall also be followed.
9 Establishment of cell lines
9.1 Processes
For establishing human PSC and mouse PSC lines, ISO 21709:2020, 5.1, shall be followed. Examples of
suitable methods for the establishment and culture of PCS lines are given in Annex B.
The biobank shall establish, implement, validate, document and maintain procedures for isolation and
primary culture of relevant cell lines.
Processes should be performed in a biosafety cabinet or under a laminar flow hood using appropriate
aseptic techniques.
For iPSC line establishment, the reprogramming strategies including reprogramming factors and gene
delivery system shall be clearly documented.
Each culture expansion is referred to as a “subculture” or “passage”.
9.2 Unique identification
The unique identification of PSC lines shall be established in accordance with ISO 20387:2018, 7.5.
®1)
This should include a unique cell line name (such as that generated by registration in the hPSCreg
[13]
database for human PSCs) or sample number, a biobank batch number and biobank vial number. Cell
lines should be anonymized or de-identified.
9.3 Testing for infectious agents
The human donor biological material or the cell line(s) derived from this material should be tested for
relevant transmittable infectious agents, e.g. HIV, HBV, HCV, HTLV, HCMV and TP. A report regarding
the condition of the mouse donor, including information on results of specific pathogen testing, shall be
obtained from the provider.
The analytical data and results as well as the associated analyses shall be documented and available
to authorized biobank personnel and researchers who process biological material and established cell
lines. ®
1) hPSCreg is an example of a suitable product available commercially. This information is given for the
convenience of users of this document and does not constitute an endorsement by ISO of this product.
10 Characterization
10.1 General
The biobank shall establish, document and implement procedures to characterize PSCs and report the
relevant data so that users can determine suitability for their intended use.
Biological characteristics of PSC lines shall be established by the biobank in accordance with current
scientific best practice and international consensus (e.g. Reference [14]). The characterization shall
include but is not limited to the following:
a) Cell morphology: Cells grow under 2D conditions and typically exhibit growth as colonies with
clear colony boundaries, high nuclear-cytoplasmic ratio and uniform morphology. Within the clone,
cell-cell contact is tight.
b) Cell identification: Cell lines have a unique donor genetic profile and this profile can be used
to facilitate exclusion of cross-contamination with other cells and confirm donor origin. The
2)
requirements on the cell line authentication can refer to ISO/TS 23511 . Biological source materials
for establishing PSC lines or the PSC in early passage shall be used where possible to establish the
initial STR profile as a reference for subsequent STR profiling, e.g. to check for cross-contamination
or cell identity.
c) Genetic integrity: Chromosome karyotype analysis: For cells from non-disease affected donors,
these are typically as follows: human PSCs: 46, XX or 46, XY; mouse PSCs: 40, XX or 40, XY.
For biobanks of PSC lines, karyological variants can arise and in such cases the potential impact on
cell line performance should be assessed either by the biobank or by users of the biobank.
d) Cell viability: A range of viability assays can be used and each measures a different aspect of cell
biology. Such tests include cell metabolic activity [the function of esterase, Thiazole blue method
based on the determination of succinic dehydrogenase (MTT also known as 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide), apoptosis markers, cell redox potential, membrane
potential, proliferation rate (DNA content), mitochondrial function and membrane integrity].
NOTE 1 Cell viability is addressed in ISO 23033:2021, 7.5.
e) Stem cell markers: PSCs express cell markers including but not limited to self-renewal markers
(e.g. OCT4, SOX2, NANOG in both human PSCs and mouse PSCs) and canonical markers indicative
of pluripotency (e.g. human PSC: TRA-1-81, TRA-1-60, SSEA3, SSEA4; mouse PSC: SSEA1). The
expression level of these markers can vary in different culture conditions. The stated markers shall
be present in the majority of the cells, e.g. by immunofluorescence and/or gene expression analysis.
The operator should establish cut-off values fit for purpose.
f) Pluripotency assay: A range of assays can be employed to reveal the pluripotency of PSCs (see
Table 1):
1) in vitro differentiation: induction of embryoid bodies or directed differentiation to the cell
types representative of each germ layer.
NOTE 2 Some cell lines, while failing the test, can be useful, e.g. for research to understand the
defect and for preparation of a particular type of differentiated cells.
2) Teratoma formation: Assessing the spontaneous generation of differentiated tissues from the
three germ layers following the injection of PSCs into immune-compromised mice.
NOTE 3 Some cells with incomplete pluripotency potential can also generate masses that
superficially resemble teratomas yet lack terminal three-germ-layer differentiation, potentially leading
to misinterpretation.
2) Under preparation. Stage at the time of publication: ISO/DTS 23511.2:2022.
NOTE 4 The teratoma assay is currently widely used and is considered as the gold standard
functional assay for assessing human PSC developmental potential. Due to ethical and legal
restrictions, other human PSC pluripotency assays in vivo (such as chimaera formation and tetraploid
complementation) cannot be performed.
NOTE 5 Teratomas are not generated from single cells, the teratoma assay assesses
developmental potency at a population-based level.
3) for mouse cell lines, chimaera formation: assessing whether cells can re-enter development
when introduced into host embryos at either morula or blastocyst stages.
NOTE 6 Human xenogenics are outside of the scope of this document.
NOTE 7 PSCs of high quality can support normal development and generate high-grade
chimaeras with extensive colonization of all embryonic tissues including the germ line, whereas less-
potent PSCs produce either low chimaerism or reduced embryo viability.
4) Germline transmission: assessing whether the test PSCs can generate functional gametes by
breeding chimaeras to produce all-donor PSC-derived offspring.
NOTE 8 This is prohibited in human PSC pluripotency assessing, see the international stem cell
[10]
research guidelines .
5) Tetraploid complementation: assessing whether the test PSCs can direct development of
an entire organism by introducing donor PSCs into tetraploid (4n) host blastocysts, which
can be generated by electrofusion of blastomeres at the two cell stage. The 4n blastocysts
cannot sustain normal embryonic development beyond mid-gestation, while tetraploid extra-
embryonic tissues develop normally and support donor cells. Any resulting embryos are
derived essentially entirely from donor PSCs. The ability to perform such tests can be restricted
in certain countries varies and, where not feasible, alternative validated in vitro assays can be
used.
NOTE 9 Tetraploid complementation is prohibited in h
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