ISO 18162:2024
(Main)Biotechnology — Biobanking — Requirements for human neural stem cells derived from pluripotent stem cells
Biotechnology — Biobanking — Requirements for human neural stem cells derived from pluripotent stem cells
This document specifies requirements for the biobanking of human neural stem cells (hPSC-NSCs) derived from human pluripotent stem cells (hPSCs), including the requirements for the differentiaton, culture, characterization, quality control (QC), storage, thawing and transport of hPSC-NSCs. 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 hPSC-NSCs used for research and development in the life sciences. This document does not apply to hPSC-NSCs 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.
Biotechnologie — Biobanque — Exigences relatives aux cellules souches neuronales humaines dérivées de cellules souches pluripotentes
General Information
Standards Content (Sample)
International
Standard
ISO 18162
First edition
Biotechnology — Biobanking —
2024-12
Requirements for human neural
stem cells derived from pluripotent
stem cells
Biotechnologie — Biobanque — Exigences relatives aux cellules
souches neuronales humaines dérivées de cellules souches
pluripotentes
Reference number
© ISO 2024
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ii
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviations . 5
5 General requirements . 6
5.1 General .6
5.2 Legal and ethical requirements .7
5.3 Personnel, facilities and equipment .7
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 to the establishment,
characterization and QC of hPSCs . 8
7 Generation of hPSC-NSCs from hPSCs . 8
7.1 Processes .8
7.2 Unique identification .8
7.3 Testing for infectious agents .8
7.4 Generation of hNSCs and culture .8
7.5 Subculture and limited expansion .9
8 Characterization of hPSC-NSCs . 9
8.1 General .9
8.2 Viability .10
8.3 Morphology . .10
8.4 Population doubling time and subculture/passage.11
8.4.1 PDT .11
8.4.2 Subculture/passage .11
8.5 Cell population purity .11
8.6 Proliferation .11
8.7 Differentiation capability — in vitro multi-cell type differentiation . 12
8.7.1 General . 12
8.7.2 In vitro neuronal and glial differentiation . 12
8.8 Immunophenotyping by flow cytometry . 12
8.9 Microbial contamination . 13
8.10 Neural replacement and functional recovery . 13
9 Quality control . 14
10 Storage . 14
11 Thawing .15
12 Disposal . 16
13 Distribution of hPSC-NSCs — Information for users .16
14 Transport of hPSC-NSCs . 17
14.1 General .17
14.2 hPSC-NSCs frozen in ampoules or cryovials .17
14.3 Living hNSC cultures .17
Annex A (informative) Exemplary quality control test procedure for biobanking of hPSC-NSCs . 19
Annex B (informative) Examplary methods for the generation of hNSC from hPSCs .20
Bibliography .24
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
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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
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This document was prepared by 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
Neural stem cells (NSCs) are the adult stem cells in the central nervous system (CNS). NSCs have self-
renewal ability and multipotency. NSCs can differentiate into various neurons and glial cells including
astrocytes and oligodendrocytes. NSCs play a major role in embryonic development and adult neurogenesis.
According to the hypothesis by Alvarez-Buylla, there are several types of cells can be called NSCs, including
[1]
neuroepithelium – epithelial cells of the ventricular zone (VZ) of the neural tube , radial glial cells (RGCs)
[2-4]
and basal (intermediate) progenitor cell (IPC) . In the adult, NSCs are restricted to specific brain regions,
such as the subventricular zone (SVZ) of the lateral ventricles and the subgranular zone (SGZ) of the dentate
[5]
gyrus (DG) of the hippocampus .
NOTE The term "neural" refers to any type of nerve cell, including a mixture of brain cells, whereas "neuronal" is
specifically related to neurons.
Despite these advances, substantial ambiguities persist regarding the nomenclature, nature, identity,
function, mode of isolation and experimental handling of these cells. NSCs are not fully defined by the initial
minimal criteria proposed out, and as such require careful characterization by a matrix of functional assays.
NSCs have been isolated from human fetal CNS (brain or spinal cord), cerebrospinal fluid, biopsy and autopsy
material, or differentiated from pluripotent stem cells (PSCs), which are widely used for animal and clinical
[6]
research . NSCs generated from different sources or differentiation protocols have different properties.
Different institutions use different practices for isolating, processing and biobanking these NSCs, making it
difficult to compare data and results across institutions. Thus, there is a need for standardized approaches
to isolate, process, expand and cryopreserve these NSCs.
The aim of this document is to provide general guidance for biobanking of human NSCs derived from
pluripotent stem cells (hPSC-NSCs) for research purposes. This document is applicable for academic centers,
public and private institutions performing a biobanking service of hPSC-NSCs for R&D (Research and
Development) and preclinical studies, not for clinical use.
Importantly, this document is focused on hPSC-NSCs that have been isolated, manipulated and/or propagated
from hPSCs in culture for research purposes.
v
International Standard ISO 18162:2024(en)
Biotechnology — Biobanking — Requirements for human
neural stem cells derived from pluripotent stem cells
1 Scope
This document specifies requirements for the biobanking of human neural stem cells (hPSC-NSCs) derived
from human pluripotent stem cells (hPSCs), including the requirements for the differentiaton, culture,
characterization, quality control (QC), storage, thawing and transport of hPSC-NSCs.
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 hPSC-NSCs used for research and
development in the life sciences.
This document does not apply to hPSC-NSCs 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 20387:2018, Biotechnology — Biobanking — General requirements for biobanking
ISO 21709:2020, Biotechnology — Biobanking — Process and quality requirements for establishment,
maintenance and characterization of mammalian cell lines
ISO 24603:2022, Biotechnology — Biobanking — Requirements for human and mouse pluripotent stem cells
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 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
authenticity
quality of being genuine or true
[SOURCE: ISO/TS 22859:2022, 3.1]
3.2
biobank
legal entity or part of a legal entity that performs biobanking (3.3)
[SOURCE: ISO 20387:2018, 3.5]
3.3
biobanking
process of acquisitioning and storing, together with some or all of the activities related to collection,
preparation, preservation, testing, analyzing and distributing defined biological material as well as related
information and data
[SOURCE: ISO 20387:2018, 3.6]
3.4
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 Guide 73) 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, 3.17]
3.5
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]
3.6
cell master file
complete dossier of all procedures and records used to generate cells
[SOURCE: ISO/TS 22859:2022, 3.6]
3.7
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]
3.8
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]
3.9
cryopreservation
process by which cells are maintained frozen at an ultra-low temperature in an inactive state so that they
can be revived at a later time
[SOURCE: ISO 21709:2020/Amd.1, 3.6]
3.10
differentiation
process to bring the stem cells into a defined cell state/fate
[SOURCE: ISO/TS 22859:2022, 3.11]
3.11
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]
3.12
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]
3.13
human neural stem cells derived from pluripotent stem cells
hPSC-NSCs
immature cellular population differentiated from pluripotent stem cells, which has the ability for self-
renewal and differentiation to neurons and glia cells (astrocytes or oligodendrocyte) in vitro and in vivo.
Note 1 to entry: Without any manipulation, culture-adapted hNSCs (human neural stem cells) is an alternate term
used to denote cells that are different from cells that are found in vivo. It is increasingly clear that these cell types have
different properties in terms of gene expression, functionality and phenotype.
3.14
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]
3.15
multipotent cells
cells that have the ability to differentiate into more than one, but a limited number of related cell types
3.16
passage
subculture
process of further culturing of cells in a new culture vessel to provide higher surface area/volume for the
cells to grow
[SOURCE: ISO/TS 22859:2022, 3.18]
3.17
passage number
number of subculturings 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 has been added.]
3.18
doubling time
PDT
population 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” have been added as the
preferred term and Note 1 to entry has been added.]
3.19
primary cells
cells isolated directly from body fluid, tissue or organs taken directly from an organism, using enzymatic or
mechanical methods
[SOURCE: ISO 21709:2020, 3.15, modified — "body fluid" added to definition.]
3.20
primary culture
initial in vitro cultivation of primary cells (3.19)
3.21
primary human neural stem cells derived from pluripotent stem cells
primary hPSC-NSCs
initial neural stem cells (NSCs) derived from in vitro human pluripotent stem cell (hPSC) differentiation
3.22
proliferation
cell number expansion by cell division
[SOURCE: ISO/TS 22859:2022, 3.22]
3.23
self-renewal
ability of stem cells (3.24) to divide symmetrically, forming two identical daughter stem cells
Note 1 to entry: Adult stem cells like neural stem cell, bone marrow stem cell etc. can also divide asymmetrically to
form one daughter cell which can proceed irreversibly to a differentiated cell lineage and ultimately lead to focused
functional differentiated cells, whilst the other daughter cell still retains the characteristics of the parental stem cell.
[SOURCE: ISO/TS 22859:2022, 3.23]
3.24
stem cell
non-specialized cells with the capacity for self-renewal (3.23) and differentiation potential (3.11), which can
differentiate into one or more different types of specialized cells
Note 1 to entry: Most adult stem cells are multipotent stem cells.
[SOURCE: ISO/TS 22859:2022, 3.24]
3.25
viability
attribute of being alive (e.g., metabolically active, capable of reproducing, have intact cell membrane, or have
the capacity to resume these functions) as defined based on the intended use
[SOURCE: ISO 21709:2020, 3.17]
3.26
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]
4 Abbreviations
Abbreviation Term
AD Alzheimer's disease
ALS amyotrophic lateral sclerosis
The most cited neural cell culture supplement, which is an optimized serum-free
B27
supplement used to support the neural cell culture
BDNF brain-derived neurotrophic factor
bFGF basic fibroblast growth factor
BMP bone morphogenetic protein
CFSE carboxyfluorescein succinimidyl ester
CNS central nervous system
CORIN atrial natriuretic peptide-converting enzyme
DCX doublecortin
DG dentate gyrus
DMEM/F-12 Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12
EB embryoid body
EDTA ethylene diamine tetraacetic acid
EGF epidermal growth factor
ELISA enzyme-linked immunosorbent assay
FACS fluorescence-activated cell sorting
FBS fetal bovine serum
FL fluorescence spectrophotometer
FOXA2 forkhead box protein A2
GABA gamma-aminobutyric acid
GPCs GABA-ergic progenitor cells
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCsAg hepatitis C surface antigen
HCV hepatitis C virus
HIV human immunodeficiency virus
hPSC-NSCs hPSC derived NSCs
hPSC pluripotent stem cell
IPC intermediate progenitor cell
TM a)
KOSR KnockOut Serum Replacement
Lif leukemia inhibitor factor
MNs motor nuerons
a) TM
KnockOut Serum Replacement is a trademark of ThermoFisher Scientific. This information is given for the convenience of
users of this document and does not constitute an endorsement by ISO of the product named.
b)
Neurobasal™ media is a trademark of ThermoFisher Scientific. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of the product named.
Abbreviation Term
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
N-2 Supplement is a chemically defined, serum-free supplement, which is intended
N2
TM b)
for use with Neurobasal media
NESTIN neuroepithelial stem cell protein
NeuN neuronal Nuclei
NIM neural induction medium
NPM neural proliferation media
NSC neural stem cell
PAX6 paired box 6
PBS phosphate-buffered saline
qPCR quantitative polymerase chain reaction
RGCs radial glial cells
RT-PCR real time polymerase chain reaction
SGZ subgranular zone
SOX2 Sry-related HMG box 2
SOX1 Sry-related HMG box 1
SVZ subventricular zone
TGFb transforming growth factor-beta
TP treponema pallidum
TPPS transferrin putrescine, progesterone sodium selenite
TUBB3 neuron-specific class III β-tubulin
LGE lateral ganglionic eminence
a) TM
KnockOut Serum Replacement is a trademark of ThermoFisher Scientific. This information is given for the convenience of
users of this document and does not constitute an endorsement by ISO of the product named.
b)
Neurobasal™ media is a trademark of ThermoFisher Scientific. This information is given for the convenience of users of this
document and does not constitute an endorsement by ISO of the product named.
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.
ISO 24603 shall be followed for human biological source material.
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-NSCs.
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-NSCs according to ISO 20387:2018, 7.9.2 and 7.9.3 at all stages of the biological material life cycle
(ISO 20387:2018, 3.29).
According to the characteristics of hPSC-NSCs, procedures, quality control (QC) documents for collection,
separation, expansion, storage, transportation and testing, and data analysis shall be established,
documented, implemented, regularly reviewed and updated.
The initial cell state of PSCs as well as culture methods can influence the properties of hPSC-NSCs. Workflow
steps cannot always be controlled. Thus, their impact on the hPSC-NSCs properties for biobanking shall be
investigated, and mitigation measures shall be established to enable the required quality control. In these
cases, risk assessment is recommended.
The biobank shall assess biorisks of hPSC-NSCs at the facilities and implement appropriate biosafety
measures for the protection of personnel and environment.
The authenticity and properties of hPSC-NSCs shall be monitored throughout the complete biobanking
process from generation to distribution.
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 and ISO 24603:2022, 5.2 shall be
followed.
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.
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 tissues.
5.3 Personnel, facilities and equipment
ISO 20387:2018, Clause 6, and ISO 21709:2020, 4.3, 4.4, 4.7 shall be followed.
The biobank personnel shall be appropriately and specifically trained in hPSC-NSCs generation,
characterization, culture, cryopreservation, recovery and transport.
The biobank shall ensure that external operators providing hPSC-NSCs services demonstrate relevant
knowledge, experience and corresponding skills.
The biobank shall ensure that facilities, equipment and environmental conditions do not adversely affect
hPSC-NSCs 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-NSCs and the need for aseptic processing.
5.4 Reagents, consumables and other supplies
ISO 21709:2020, 4.5 and ISO 24603:2022, 5.4 shall be followed.
The biobank shall establish acceptance criteria for materials, including reagents and consumables,
necessary for hPSC-NSCs differentiation, culture, establishment, expansion, preservation, storage, thawing
and transport.
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 hPSC-NSCs, 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.
Certain data retention times, data integrity and security of data storage shall be ensured.
For hPSC-NSCs, 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 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 to the establishment,
characterization and QC of hPSCs
ISO 24603 shall be followed.
7 Generation of hPSC-NSCs from hPSCs
7.1 Processes
For establishing hPSC-NSCs ISO 21709:2020, 5.1 shall be followed.
The biobank shall establish, implement, validate, document and maintain procedures for hPSC-NSCs
generation from hPSCs.
Processes should be performed in a biosafety cabinet or under a laminar flow hood using appropriate aseptic
techniques.
Each culture expansion is referred to as a ”subculture” or “passage”.
7.2 Unique identification
The unique identification of hPSC-NSCs shall be established in accordance with ISO 20387:2018, 7.5. This
should include a unique cell name or sample number, a biobank batch number and biobank vial number. Cells
should be anonymized or de-identified.
7.3 Testing for infectious agents
The starting hPSCs should be tested for relevant transmittable 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 hNSCs and culture
hPSC-NSCs can be generated from hPSCs by methods combining set of differentiation-inducing factors (e.g.,
growth factors, low-molecular weight compounds, oxygen concentration during cell culture) and cell culture
condition (e.g., suspension, monolayer). The dual SMAD inhibition (inhibition of bone morphogenetic protein
(BMP) and transforming growth factor-beta (TGFb) signaling) is widely used for inducing differentiation of
[7]
hPSCs . Examples of suitable methods for the differentiation and culture of hPSC-NSCs are given in Annex B
and references [8, 9].
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 neural stem cells from pluripotent stem cells, the differentiation strategies shall be clearly
documented.
NOTE 1 The self-renewal of hPSC-NSCs depends on the presence of epidermal growth factor (EGF) and basic
fibroblast growth factor (bFGF).
NOTE 2 Long-term maintenance of hPSC-NSCs in vitro remains a challenge, because it can cause change in the
characteristics of hPSC-NSCs, including but not limited to:
[9]
a) tendency to differentiate into specific region of brain, e.g., forebrain to mid/hindbrain
b) population doubling time (both decreasing and increasing can happen);
c) ability to differentiate into neuron;
[8,10]
d) genetic instability (karyotype abnormality) .
7.5 Subculture and limited expansion
A culture can be further expanded for biobanking after successful establishment of the primary hPSC-NSCs;
this is then known as a subculture. Each culture expansion is referred to as a "subculture" or "passage".
Cultures should be tested for microbiological contaminants (including bacteria, fungi, mycoplasma,
endotoxins and adventitious viral agents) before any further expansion.
Cell passaging follows the relative protocols after establishment of primary culture. Expansion of hPSC-
NSCs is recommended for up to 3 passages 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., undifferentiation status and
immunophenotyping).
During hNSC culture and expansion it is important that the cells do not differentiate prematurely. This can
[11]
have an unfavorable effect on product quality and yield .
8 Characterization of hPSC-NSCs
8.1 General
The biobank shall establish, document and implement procedures to characterize hPSC-NSCs 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-NSCs in culture. The characterization shall
include, but is not limited to:
a) authentication;
b) cell morphology;
c) growth kinetics;
d) viability;
e) differentiation capability in vitro;
f) immunophenotype;
g) functional characterization in vitro;
h) being free of microbial contamination;
i) karyotyping.
Exemplary methods for hPSC-NSCs characterization tests can be found in Annex B.
8.2 Viability
The biobank shall define, implement and document a procedure to determine cell viability.
Quality control for cell viability test shall be performed using live and dead cells. Cell viability shall be
determined and documented.
The biobank shall assess the amount of viable cells in the cell culture at regular intervals and especially
after changes of cell culture conditions.
Viability shall be assessed following recovery from cryopreservation.
The biobank shall limit the acceptable percentage of nonviable cells in the population during the test
procedure.
The amount of viable hPSC-NSCs should be ≥ 80 % prior to cryopreservation.
The amount of viable hPSC-NSCs should be ≥ 60 % immediately post-thaw and determined with a
validated method.
NOTE A viability assay is usually performed following 24 h to 48 h in culture after recovery to avoid false
overestimation of viability.
An automated cell viability test should be performed.
The biobank can define, implement and document a procedure to evaluate apoptosis in the cell culture.
EXAMPLE Assessment on any cryopreserved and thawed hPSC-NSCs for apoptosis is typically done using flow
cytometry (see B.7).
8.3 Morphology
Cell morphology can be very different depending on growth conditions and different differentiation
strategies. In addition, at different stages of culture cells have different morphologies reflecting changes in
status, such as generation of EB (embryoid body) followed by differentiation into neural rosettes. EBs are 3D
spherical aggregates that recapitulate several aspects of early embryogenesis.
A description of cell morphology should include the conditions of culture as well as the stages of culture.
NOTE hPSC-NSCs can be maintained, and possibly expanded, without adherence under specific culture conditions.
But these cells, if maintained under more standardized conditions, would be expected to demonstrate adherence.
The biobank shall document significant cell morphology changes.
8.4 Population doubling time and subculture/passage
8.4.1 PDT
The PDT is the time (measured in hours) required for the replication of the population of hPSC-NSCs. 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 PDT.
NOTE 1 Formula (1) is applicable in a linear range of cell expansion.
The average PDT of hPSC-NSCs isolated from hPSCs ranges between 20 h and 40 h.
NOTE 2 Depending on the culture conditions, culture passage, cell density and characteristics of the donor (e.g.,
age), the PDT can vary.
The PDT of hPSC-NSCs should be determined by the biobank after secondary culture.
PDT can reflect the growth kinetics of hPSC-NSCs in culture. The biobank can utilize the PDT of hNSC
cultures at different passages to evaluate changes in culture cell growth kinetics.
The PDT shall be documented.
8.4.2 Subculture/passage
P , the passage number(s) together with the seeding and final cell density, and the culture vessel surface
area shall be documented. For 2D culture, when the hPSC-NSCs cover the culture vessel at 80 % to 90 %, the
cells can be passaged.
Passage numbers are frequently used by laboratories. However, passage number is correlated with the
surface area/volume of a culture vessel and how the initial P is defined. It is recommended that the biobank
defines P as the initial plating of hPSC-NSCs.
Documenting PDT along with passage numbers can facilitate a better understanding of growth dynamics of
the hPSC-NSCs and the relationship between passages and PDT.
8.5 Cell population purity
The biobank shall evaluate the purity of hPSC-NSCs and unwanted cell populations such as astrocyte, neuron
and other mature neural cells should be < 10 %. Immunophenotyping of hPSC-NSCs as described in 8.8 can
be used for evaluating and verifying purity and identity. Unwanted microbial contaminants shall be defined
and checked, as described in 8.9.
NOTE Since definitive markers for neural stem cells have not been established, purity can vary depending on the
markers used for evaluation.
8.6 Proliferation
The biobank shall define, implement and document a cell proliferation assay.
The assay for cell proliferation shall be documented including internal QC criteria as described in Clause 8.
NOTE Cell proliferation can be evaluated by CFSE or by MTT (see B.5 and B.6).
8.7 Differentiation capability — in vitro multi-cell type differentiation
8.7.1 General
To evaluate in vitro multilineage differentiation of hPSC-NSCs, the biobank should establish, document and
implement procedures and requirements for the specific assays, (i.g., hPSC-NSCs with ability to undergo in
vitro multi-cell type differentiation). The following in vitro multi-cell type differentiation assays are part of
the in vitro characterization of hPSC-NSCs, but do not always reflect the in vivo differentiation capacity of
these cells.
In vitro multi-cell type differentiation into neuron and astrocyte under appropriate conditions is a valuable
tool for characterization of hPSC-NSCs. This information can be useful, if assessed in a quantitative assay
format that is validated and sufficiently sensitive to assess hPSC-NSCs grown for different lengths of time,
and under different conditions.
8.7.2 In vitro neuronal and glial differentiation
hPSC-NSCs can differentiate into various types of neurons and astrocytes in different induction medium.
hNSCs can differentiate into motor neurons, dopaminergic neurons and other types of neurons as well
as various types of glial cells. For cells at these stages of development, there are several protocols for
differentiation, see in Annex B.
NOTE Typical neural induction medium in general contains DMEM/F12, N2, B27, L-Glutamine and other cytokines.
8.8 Immunophenotyping by flow cytometry
NOTE Cell surface marker expression has been described for the identification and isolation of many neural cell
types by fluorescence-activated cell sorting (FACS) from embryonic and adult tissue from multiple species. hPSC-NSCs
[12,13]
have been isolated from human brain using genetic promoter-reporters of hPSC-NSCs markers . hPSC-NSCs are
characterized by the presence of specific cell markers and the absence of others, for example, hPSC-NSCs from neural
[14]
induction cultures of hPSCs are SOX1+/SOX2+/NESTIN+/CD133+/OCT4- .
[15]
CD133 has proven useful for isolating hNSC in multiple studies . However, due to its high expression in
hPSCs and dim expression in EB-rosette(+) cultures, SOX2 or CD133 should be avoided as a selection marker
[14][16]
for hPSC-NSCs, which reduces the risk of insufficient purification away from hPSCs .
hPSC-NSCs can be characterized by a
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