ISO/FDIS 20012

FINAL DRAFT
International
Standard
ISO/TC 276
Biotechnology — Biobanking —
Secretariat: DIN
Requirements for human natural
Voting begins on:
killer cells derived from pluripotent
2025-11-18
stem cells
Voting terminates on:
2026-01-13
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
IN ADDITION TO THEIR EVALUATION AS
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
Reference number
FINAL DRAFT
International
Standard
ISO/TC 276
Biotechnology — Biobanking —
Secretariat: DIN
Requirements for human natural
Voting begins on:
killer cells derived from pluripotent
stem cells
Voting terminates on:
RECIPIENTS OF THIS DRAFT ARE INVITED TO SUBMIT,
WITH THEIR COMMENTS, NOTIFICATION OF ANY
RELEVANT PATENT RIGHTS OF WHICH THEY ARE AWARE
AND TO PROVIDE SUPPOR TING DOCUMENTATION.
© ISO 2025
IN ADDITION TO THEIR EVALUATION AS
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
BEING ACCEPTABLE FOR INDUSTRIAL, TECHNO­
LOGICAL, COMMERCIAL AND USER PURPOSES, DRAFT
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting on
INTERNATIONAL STANDARDS MAY ON OCCASION HAVE
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TO BE CONSIDERED IN THE LIGHT OF THEIR POTENTIAL
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TO BECOME STAN DARDS TO WHICH REFERENCE MAY BE
MADE IN NATIONAL REGULATIONS.
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Published in Switzerland Reference number
ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviations . 4
5 General requirements . 5
5.1 General .5
5.2 Legal and ethical requirements .5
5.3 Personnel, facilities and equipment .5
5.4 Reagents, consumables and other supplies .5
5.5 Management of information and data .6
6 hPSCs and associated data . 6
7 Generation of hPSC-NK cells from hPSCs . 6
7.1 Processes .6
7.2 Unique identification .7
7.3 Testing for infectious agents .7
7.4 Generation of hPSC-NK cells and culture .7
7.5 Subculture and limited expansion .7
8 Characterization of hPSC-NK cells . 7
8.1 General .7
8.2 Viability .8
8.3 Morphology . .8
8.4 Population doubling time (PDT) and subculture/expansion .8
8.4.1 PDT .8
8.4.2 Expansion .9
8.5 Cell population purity .9
8.6 Immunophenotyping by flow cytometry .9
8.7 Microbiological contamination .10
8.8 hPSC-NK cell activity and function . .10
9 Quality control .11
10 Storage .11
11 Thawing and culturing .12
12 Disposal .13
13 Distribution of hPSC-NK cells — Information for users .13
14 Transport of hPSC-NK cells . 14
14.1 General .14
14.2 Transport of frozen hPSC-NK cells .14
14.3 Transport of living hPSC-NK cells . . . 15
Annex A (informative) Examples of quality control tests for processing of hPSC-NK cells .16
Annex B (informative) Examples of methods for the generation of hPSC-NK cells from hPSCs . 17
Annex C (informative) Example of a quality control test procedure for hPSC-NK cells . 19
Bibliography .21

iii
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 document 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).
ISO draws attention to the possibility that the implementation of this document may involve the use of (a)
patent(s). ISO takes no position concerning the evidence, validity or applicability of any claimed patent
rights in respect thereof. As of the date of publication of this document, ISO had not received notice of (a)
patent(s) which may be required to implement this document. However, implementers are cautioned that
this may not represent the latest information, which may be obtained from the patent database available at
www.iso.org/patents. ISO shall not be held responsible for identifying any or all such patent rights.
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.

iv
Introduction
Natural killer (NK) cells, also known as large granular lymphocytes (LGL), are a type of cytotoxic
lymphocytes belonging to the innate immune system. NK cells constitute 5 % to 15 % of the mononuclear
[1]
cells and up to 20 % of lymphocytic population in the peripheral blood. The expression patterns of
activating receptors and inhibitory receptors determine the activating status and functionalities of NK
cells. NK cells can eliminate pathogen-infected cells, cancerous cells, and other unhealthy cells by direct
cytotoxicity. They express apoptosis-related ligands (TRAIL and FasL) and cytotoxic granules (granzymes
and perforin) which can induce cell death in stressed cells. NK cells with the expression of CD16 can also
kill antibody-coated target cells by antibody-dependent cell cytotoxicity (ADCC). As innate immune cells,
NK cells are also engaged in reciprocal interactions with other immune cells to limit or exacerbate immune
[2]
responses .
In adult, hematopoietic stem cells (HSCs) differentiate into common lymphoid progenitors (CLPs), then,
give rise to NK progenitors (NKP). NKPs migrate into all lymphoid tissues or organs, and differentiate
[3]
into mature NK cells subsequently . However, NK cells can also arise from erythro-myeloid progenitors
[4]
(EMPs) in the yolk sac. NK cells can be generated by induction from human pluripotent stem cells (hPSCs) .
Regarding the various tissue origins of natural NK cells during development, there also has been established
various methods for generating induced NK cells from hPSCs. In the NK cell regeneration system, hPSCs are
first induced into mesoderm progenitors or lateral plate mesoderm cells via embryo body (EB) formation or
monolayer differentiation methods. Then, these cells can be induced into hemogenic endothelial cells (HECs)
which further differentiate into hematopoietic progenitor cells (HPCs) via hematopoietic transition (EHT).
[5] [6]
NK cells can even totally regenerated from these HPCs under specified cytokine combinations , .
NK cells are fragile and sensitive to cryopreservation and thawing. Standardized approaches to maintain
the functionality of banked NK cells is needed. hPSC-dervied NK cell are primary cells, not immortalized,
and therefore have a finite life span.
This document is applicable for academic centers, public and private institutions performing NK cell
generation from hPSCs (Research and Development) and preclinical studies, not for clinical use.
Importantly, this document is focused on NK cell regeneration that have been reported from hPSCs in culture
for research purposes.
EXAMPLE Applications of NK cells (e.g. immunoresponse regulation, anti-tumor or anti-viral)

v
FINAL DRAFT International Standard ISO/FDIS 20012:2025(en)
Biotechnology — Biobanking — Requirements for human
natural killer cells derived from pluripotent stem cells
1 Scope
This document specifies requirements for the biobanking of human natural killer (NK) cells derived
from human pluripotent stem cells (hPSCs), including the requirements for the differentiaton, culture,
characterization, quality control, storage, thawing and transport of NK cells.
Requirements for the collection of biological source material, the transport to and reception of biological
source material and hPSCs at the biobank, as well as the establishment, expansion and QC of hPSCs are
covered in ISO 24603.
This document is applicable to all organizations performing biobanking of human NK cells used for research
and development in the life sciences.
This document does not apply to human NK cells for the purpose of 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, Biotechnology — Biobanking — General requirements for biobanks
ISO 21709, Biotechnology — Biobanking — Process and quality requirements for establishment, maintenance
and characterization of mammalian cell lines
ISO/TS 23511, Biotechnology — General requirements and considerations for cell line authentication
ISO 24603, Biotechnology — Biobanking — Requirements for human and mouse pluripotent stem cells
ISO 24190, Biotechnology — Analytical methods — Risk-based approach for method selection and validation for
rapid microbial detection in bioprocesses
ISO 35001, Biorisk management for laboratories and other related organisations
ISO 35001:2019/Amd 1:2024, Biorisk management for laboratories and other related organisations —
Amendment 1: Climate action changes
ISO 8934-1, Cell viability analytical methods — Part 1: General requirements and considerations
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 20387, ISO 21709 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
authenticity
quality of being genuine or true
[SOURCE: ISO/TS 22859:2022, 3.1]
3.2
biorisk
effect of uncertainty expressed by the combination of the consequences of an event (including changes in
circumstances) and the associated “likelihood” (as defined in ISO 31073) of occurrence, where biological
material is the source of harm
Note 1 to entry: The harm can be the consequence of an unintentional exposure, accidental release or loss, theft,
misuse, diversion, unauthorized access or intentional unauthorized release.
[SOURCE: ISO 35001:2019, 3.16]
3.3
cell culture
growth of cells dissociated from the parent tissue by spontaneous migration, mechanical or enzymatic
dispersal for propagation under in vitro conditions
[SOURCE: ISO/TS 22859:2022, 3.5]
3.4
cell master file
complete dossier of all procedures and records used to generate cells
[SOURCE: ISO/TS 22859:2022, 3.6]
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 specific markers, genetic polymorphisms and biological activities
[SOURCE: ISO/TS 22859:2022, 3.8]
3.6
cryopreservation
process by which cells are maintained frozen at 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 stem cells into a defined cell state or fate
[SOURCE: ISO/TS 22859:2022, 3.11]

3.8
flow cytometry
methodologically oriented subdiscipline of analytical cytology that measures cells in suspension in a liquid
vehicle as they pass, typically one cell at a time, by a measurement station
Note 1 to entry: The measurement represents transformations of changes in the output of a detector (or detectors)
due to changes in scattered light, absorbed light, light emitted (fluorescence) by the cell, or changes in electrical
impedance, as the cell passes through the measuring station.
Note 2 to entry: Flow cytometry allows simultaneous evaluation of morphological characteristics of cells (size and
internal complexity) with membrane or intracellular antigens.
[SOURCE: ISO/TS 22859:2022, 3.13]
3.9
human NK cells derived from pluripotent stem cells
hPSC-NK cells
innate lymphocytes that are differentiated from pluripotent stem cells (ESC and iPSC,
CiPSC), which have the abilities of non-specific cytotoxicity, antibody-dependent cell cytotoxicity (ADCC)
Note 1 to entry: Type of immune cell that has granules (small particles) with enzymes that can kill tumor cells or cells
infected with a virus. A natural killer cell is a type of white blood cell.
3.10
viable cells
cells within a sample that have an attribute of being alive (e.g. metabolically active, capable of reproduction,
possessed of intact cell membrane, or with the capacity to resume these functions) defined based on the
intended use
[SOURCE: ISO 20391-1:2018, 3.29]
3.11
cryoprotectant
agents that protect biological material from freezing damage
3.12
vitrification
process that involves rapidly cooling biological material to transform it into a glass-like, amorphous state
without the formation of ice crystals, thereby preserving its structure and function
3.13
cell line-derived xenograft
CDX
type of animal model used in cancer research involves implanting human cancer cell lines into
immunocompromised mice to study tumor growth and test potential cancer treatments
3.14
antibody-dependent cellular cytotoxicity
ADCC
immune response where antibodies bind to a target cell, marking it for destruction by immune cells such as
natural killer (NK) cells
4 Abbreviations
Abbreviation Term
ADCC Antibody-dependent cell cytotoxicity
bFGF Basic fibroblast growth factor
BMP4 Bone morphogenetic protein 4
CFSE Carboxyfluorescein succinimidyl ester
CiPSC Chemically induced pluripotent stem cells
CLPs Common lymphoid progenitors
DMEM/F-12 Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12
E:T Effector : Target
EB Embryoid body
EDTA Ethylene diamine tetraacetic acid
EGF Epidermal growth factor
EHT Endothelial to hematopoietic transition
EMPs Erythro-myeloid progenitors
ESC Embryonic stem cell
FACS Fluorescence-activated cell sorting
FasL Fas ligand
FBS Fetal bovine serum
HBV Hepatitis B virus
HCV Hepatitis C virus
HCMV Human Cytomegalovirus
HECs Hemogenic endothelial cells
HIV Human immunodeficiency virus
HPCs Hematopoietic progenitor cells
hPSC Human pluripotent stem cell
hPSC-NK cells hPSC derived natural killer cells
HSCs Hematopoietic stem cells
HTLV Human T-cell lymphotropic virus
IGF-1 Insulin-like Growth Factor I
iPSC Induced Pluripotent Stem Cells
LGL Large granular lymphocytes
NKP NK progenitors
PBS Phosphate-buffered saline
SCF Stem cell factor
a
SGR-SM SUPERGROW® Cell Culture Supplemental Mix
TP Treponema pallidum
TPO Thrombopoietin
TRAIL Tumor Necrosis Factor-Related Apoptosis Inducing Ligand
VEGF Vascular endothelial growth factor
a
SGR-SM is the SUPERGROW® Cell Culture Supplemental Mix of a product supplied by Dakewe Biotech Co., Ltd. This
information is given for the convenience of users of this document and does not constitute an endorsement by ISO of
the product named. Equivalent products may be used if they can be shown to lead to the same results.

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.
The biobank shall establish criteria and procedures for the establishment and expansion of PSCs,
differentiation, culture, characterization, quality control, storage, thawing and transport of hPSC-NK cells.
ISO 24603 shall be followed.
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 hPSC-
NK cells at all stages of the biological material life cycle.
According to the characteristics of hPSC-NK cells, procedures, quality control (QC) documents for expansion,
storage, transportation and testing, and data analysis shall be established, documented, implemented,
regularly reviewed and updated.
The biobank shall assess biorisks of hPSC-NK cells at the facilities and implement appropriate biosafety
measures for the protection of personnel and environment.
ISO 35001 and ISO 35001:2019/Amd 1:2024 shall be followed for biorisk assessment.
The authenticity and properties of hPSC-NK cells shall be monitored throughout the complete biobanking
process from generation to distribution. ISO/TS 23511 shall be followed.
5.2 Legal and ethical requirements
The biobank shall collect relevant information on ethical requirements, implement and regularly update
them, where relevant.
The biobank shall establish, document and implement policies on the procurement and supply of PSCs.
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 tissues.
5.3 Personnel, facilities and equipment
The biobank personnel shall be appropriately and specifically trained in hPSC-NK cell generation,
characterization, culture, cryopreservation, recovery and transport.
The biobank shall ensure that external operators providing hPSC-NK cell services demonstrate relevant
knowledge, experience and corresponding skills.
The biobank shall ensure that facilities, equipment and environmental conditions do not adversely affect
hPSC-NK cell quality 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 hPSC-NK cells and the need for aseptic processing.
5.4 Reagents, consumables and other supplies
The biobank shall establish acceptance criteria for materials, including reagents and consumables, necessary
for hPSC-NK cell differentiation, culture, expansion, preservation, storage, thawing and transport.

5.5 Management of information and data
The biobank shall manage and maintain associated data of hPSC-NK cells, including but not limited to the
following:
a) the technical information: methods used in the generation of cells, culture conditions, passage data
including passage number, characterization, microbiological test data;
b) the preservation and storage information;
c) the characterization and 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.
NOTE 1 The following biological material can be considered for testing:
— cultured hPSC-NK cells derived from hPSCs (for PCR testing to detect expression of certain gene(s).
NOTE 2 For cell identity verification methods, ISO 23511 can be used.
Certain data retention times, data integrity and security of data storage shall be ensured.
For hPSC-NK cells, 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 the relevant requirements of ISO 20387.
The cell master file shall be kept to enable review of the data and records for specific applications.
6 hPSCs and associated data
If the biobank collects the biological source material and associated data, the biobank shall follow ISO 24603
for the collection and transport of the biological source material and associated data as well as the
establishment, characterization and QC of hPSCs.
If the biobank does not collect the biological source material and associated data, but has control over the
collection, the biobank shall request that the collection and transport of biological source material and
associated data are performed in accordance with ISO 24603. The biobank shall follow ISO 24603 for the
establishment, characterization and QC of hPSCs.
EXAMPLE If the biobank requests the collection process to be performed by another party, they have control over
the requirements for the collection and transport, and thus can request specific methods, transport temperatures etc.
If the biobank acquires biological source material and associated data, ISO 24603 shall be followed for the
the establishment, characterization and QC of hPSCs.
If the biobank acquires hPSCs and associated data, the biobank shall evaluate the acquired hPSCs for fitness
for purpose and shall perform QC in accordance with ISO 20387, ISO 21709 and ISO 24603.
In any case, the followed procedures shall be documented.
7 Generation of hPSC-NK cells from hPSCs
7.1 Processes
For establishing hPSC-NK cells, the relevant requirements of ISO 21709 shall be followed.
The biobank shall establish, implement, validate, document and maintain procedures for hPSC-NK cell
generation from hPSCs.
Processes should be performed in a biosafety cabinet or under a laminar flow hood using appropriate aseptic
techniques.
7.2 Unique identification
The unique identification of hPSC-NK cells shall be established. This should include a unique cell name
or sample number, a biobank batch number and biobank vial number. Cells should be anonymized or
pseudonymized.
7.3 Testing for infectious agents
Starting materials used to derive NK cells should be tested for relevant infectious agents, e.g., HIV, HBV, HCV,
HTLV, HCMV, toxoplasmosis and TP.
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 cells.
7.4 Generation of hPSC-NK cells and culture
hPSC-NK cells can be generated by inducing the differentiation of hPSCs. Examples of suitable methods for
the differentiation and culture of hPSC-NK cells are given in Annex B.
The biobank shall establish, implement, validate, document and maintain procedures for differentiation and
culture of relevant cell lines.
Processes should be performed in a biosafety cabinet or under a laminar flow hood using appropriate aseptic
techniques.
For deriving natural killer cells from pluripotent stem cells, the differentiation strategies shall be clearly
documented.
NOTE The expansion of hPSC-NK cells depends on the presence of interleukin-2.
7.5 Subculture and limited expansion
A culture can be further expanded for biobanking after successful establishment of the primary hPSC-NK
cells; this is then known as a subculture. Each culture expansion is referred to as a "subculture".
Cultures should be tested for microbiological contaminants (including bacteria, fungi, yeast, mycoplasma,
endotoxins and adventitious viral agents) before any further expansion.
Cell expansion follows the relative protocols after establishment of primary culture. Expansion of hPSC-
NK cells is recommended for up to 7 days to ensure sufficient availability of material while preserving the
biological features of the original culture, thus preventing culture-associated adaptations. The biobank
shall monitor the expansion for changes in specific biological characteristics (e.g., cytotoxic activity and
immunophenotyping).
8 Characterization of hPSC-NK cells
8.1 General
The biobank shall establish, document and implement procedures to characterize hPSC-NK cells and report
the relevant data so that users can determine suitability for their intended use.
The biobank shall define a matrix of assays and a set of markers based at least on Clause 7.

The biobank shall perform ongoing characterization of hPSC-NK cells in culture. The characterization shall
include, but is not limited to:
a) authentication;
b) cell morphology;
c) cell viability;
d) immunophenotype;
e) functional characterization in vitro;
f) being free of microbial contamination;
g) growth kinetics;
h) karyotyping.
Exemplary methods for hPSC-NK cell characterization tests can be found in Table A.1.
8.2 Viability
The biobank shall define, implement and document a procedure to determine cell viability, which is in
accordance with ISO 8934-1.
Quality control for cell viability testing shall be performed using viable and non-viable cells. Cell viability
shall be determined and documented.
The biobank shall assess the quantity of viable cells in the cell culture at regular intervals and especially
after changes of cell culture conditions.
The hPSC-NK cells should be differentiated within 24 h and the viability of these hPSC-NK cells should be ≥ 70 %.
The percentage of viable hPSC-NK cells should be determined with a validated method immediately post-
thaw and prior to application. The required percentage of viable cells varies depending on the specific
intended use.
An automated cell viability test should be performed.
The biobank can define, implement and document a procedure to evaluate apoptosis in the cell culture.
8.3 Morphology
ISO 24479 should be followed. In addition, a description of cell morphology should include the conditions of
culture as well as the status of cells.
hPSC-NK cell morphology can be different depending on different status, and these cells become larger and
irregular upon activation. Cells should be suspended in resting status. After activation, the volume becomes
larger and the cell shape becomes irregular.
The biobank shall document cell morphology changes.
8.4 Population doubling time (PDT) and subculture/expansion
8.4.1 PDT
The PDT is the time (measured in hours) required for the replication of the population of hPSC-NK cells. The
PDT is calculated with Formula (1) using the cell counts obtained before and after harvest:

D = (T -T ) × log2/ (log N - log N ) (1)
0 0
where
(T-T ) is the incubation time in hours;
N is the count of cells harvested;
N is the count of cells seeded;
D is the PDT.
NOTE 1 Formula (1) is applicable in a linear range of cell expansion.
NOTE 2 Depending on the culture conditions, differentiation stages, cell density and characteristics of the donor
(e.g., age), the PDT can vary. The average PDT of hPSC-NK cells isolated from hPSCs ranges between 24 h and 48 h.
The PDT of hPSC-NK cells should be determined by the biobank after secondary culture.
PDT can reflect the growth kinetics of hPSC-NK cells in culture. The biobank can utilize the PDT of hPSC-NK
cell cultures at different stages to evaluate changes in culture cell growth kinetics.
The PDT shall be documented.
8.4.2 Expansion
6 6
hPSC-NK cells can be usually expanded or cultured at a density of 1,5 × 10 /ml to 2 × 10 /ml. When 80 % to
90 % of the culture vessel was covered by hPSC-NK cells, the cells can be subcultured.
8.5 Cell population purity
The biobank shall evaluate the purity of hPSC-NK cells described as following:
CD56 positive > 90 %, CD45 positive > 90 %, CD16 positive > 10 %.
Unwanted cell populations such as T cells (CD3 positive) should be < 5 %.
Immunophenotyping of hPSC-NK cells as described in Annex C can be used for evaluating and verifying
purity and identity. Unwanted microbial contaminants shall be defined and checked, as described in 8.7.
hPSC-NK cells can be assessed for additional NK cell markers for specific intended purposes: FasL, TRAIL,
KIR, NKG2D, NKp44, and NKp46.
8.6 Immunophenotyping by flow cytometry
hPSC-NK cells should be characterized by a panel that includes at least the markers (antigens) and
expressions of antigens listed in Table 1. Additionally, other international guidelines should be followed,
where appropriate.
Table 1 — Antigens and clones of antibodies recommended for immunophenotyping of hPSC-NK cells
Antigen Antibody clones Required detection rate
in %
CD45 HI30 ≥ 90,0
CD56 HCD56 ≥ 90,0
CD16 3G8 ≥ 10,0
CD3 UCHT1 ≤ 5,0
NOTE 1 Cell surface marker expression has been described for the identification and isolation of natural killer cells
by fluorescence-activated cell sorting (FACS) from embryonic and adult tissues. hPSC-NK cells can be characterized by
the presence of specific cell markers and the absence of others, for example, hPSC-NK cells from human cord blood or
peripheral blood will be CD45+CD56+CD16+/-CD3-.
NOTE 2 Cells can resuspend in staining buffer. Cells can be washed with phosphate-buffered saline (PBS) (pH 7,4),
The cells can be labelled with the selected antibodies in the dark for 15 min on ice. Isotype-matched control antibodies
can be used as controls. Cells can be analyzed in a FACS flow cytometer.
Annex C includes an informative general guideline on immunophenotyping by flow cytometry.
8.7 Microbiological contamination
ISO 24190 shall be followed.
Procedures for microbiological contaminant testing of hPSC-NK cells shall be established, validated,
implemented and documented throughout the whole process.
Microbiological contaminant testing shall be performed during the processes of differentiation, culture,
subculture, cryopreservation, and after thawing. The testing methods for microbiological contamination
from ISO/TR 4752 can be used.
Throughout the whole process from donation and procurement, preparation of culture reagents and
equipment, to maintenance and cryopreservation of cultures, it is important to take a holistic view and
establish microbiological testing at all critical points of the process. In addition, procedures to minimize
risks to other established cultures should be in place. It is good practice to maintain QC procedures for
primary tissues or cells newly brought into the biobank. Such cultures should be maintained in a dedicated
area and in segregated equipment until sufficient data is available to justify their relocation.
Test methods used for microbiological testing shall be validated. It is important to be sure that appropriate
levels of sensitivity, specificity and robustness are being used in respect of testing cell cultures.
Microbial contamination shall be assessed by risk management throughout the process.
hPSC-NK cells used for research and development shall be free of contaminants. These contaminants include,
but are not limited to bacteria, yeast, fungi, and mycoplasma:
a) Tests for the presence of bacteria, yeast, fungi, and mycoplasma shall be conducted routinely. Samples
tested for the presence of bacteria, yeast, fungi and mycoplasma should be free of antibiotics or other
factors that can interfere with the accuracy of the tests.
b) It is also necessary to be aware of the impact, which some contamination can have on the biological
characteristics of the cell population being cultured. For example, a low level of viral infection will
probably not have a significant impact on cell death, but can dramatically influence the biological
activity. This type of contamination can impact any research data being generated.
EXAMPLE Mycoplasma is recognized as a common contaminant of cell cultures due to the risk of contamination
from numerous sources. Mycoplasma can be very difficult to remove from cell culture as their small size limits
filtration and they can be difficult to detect without establishing routine testing procedures.
The risks posed by transmissible spongiform encephalopathies (TSEs) should be considered irrespective of
the origin or history of the cells. There are a number of TSE diseases across the globe showing their ability of
transmitting to humans.
8.8 hPSC-NK cell activity and function
The activity and cytotoxicity function of hPSC-NK cells shall be tested both in vitro and in vivo. In vitro,
hPSC-NK cells shall express granzyme, perforin and interferon upon activation. hPSC-NK cells shall kill the
target cells whose HLA-I expression is down-regulated or lost. In addition, hPSC-NK cells shall have the
capability of antibody-dependent cellular cytotoxicity (ADCC).

In vivo, the cytotoxicity performance of hPSC-NK cells shall be tested by using cell line-derived xenograft (CDX).
NOTE The protocol as described in Annex C can be used for evaluating cell activity and function of hPSC-NK cells.
9 Quality control
The biobank shall establish, implement and document a QC procedure which shall include the testing of
biological characteristics related to the in vitro functionality of hPSC-NK cells as given in Clause 8.
QC of biological characteristics (see Clause 8) of hPSC-NK cells shall be performed for all critical procedures,
from isolation to thawing. A recommended QC procedure for biobanking of hPSC-NK cells is given in Annex C.
The biobank shall establish, implement and document QC acceptance criteria for all the biological
characteristics of hPSC-NK cells included in Clause 8.
The biobank shall establish, implement and document QC acceptance criteria for all critical control points,
e.g. culture media, reagents and equipment.
Throughout the biobanking processes, the culture media shall be periodically tested for Mycoplasma spp.
QC shall be established with a risk-based approach related to laboratory safety.
Prior to freezing, cell morphology (8.3), immunophenotyping (8.6), and viability (8.2) shall be tested and the
results shall be consistent with features of hPSC-NK cells.
10 Storage
10.1 The storage requirements of ISO 20387 and ISO 21709 shall be followed.
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NOTE hPSC-NK cells can be cryopreserved using both DMSO and FBS-free systems, comprising different
polymers either alone or in combination with ethylene glycol, 1,2-propylene glycol, trehalose (a disaccharide with
recognised neuroprotective features), sucrose, and/or glucose.
10.2 For fresh cells, hPSC-NK cells should be suspended in culture media at room temperature (15 °C to
25 °C) or in a cold storage solution (2 °C to 8 °C).
10.3 When freezing hPSC-NK cells, it is important that the cells are in the growth phase. Optimizing the
cryopreservation procedure and methods is crucial to minimize cell damage during freezing and thawing,
ensuring the availability of viable cells.
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10.4 The hPSC-NK cells should be frozen in a density between 10×10 cells/ml to 50×10 cells/ml.
10.5 For cryopreserved hPSC-NK cells, the following information shall be documented:
a) the cell name;
b) the preserved hPSC-NK cell batch number;
c) the date of preservation according to ISO 8
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