ISO 24651:2022

INTERNATIONAL ISO
STANDARD 24651
First edition
2022-08
Biotechnology — Biobanking
— Requirements for human
mesenchymal stromal cells derived
from bone marrow
Biotechnologie — Biobanking — Exigences relatives aux cellules
stromales mésenchymateuses dérivées de la moelle osseuse
Reference number
© ISO 2022
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ii
Contents Page
Foreword .v
Introduction . vi
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms and symbols .5
5 General requirements . 7
5.1 General . 7
5.2 Personnel, facilities and equipment . 7
5.3 Reagents, consumables and other supplies . 8
5.4 Management of information and data . 8
6 Collection of bone marrow samples and associated data . 8
6.1 Information about the bone marrow donor . 8
6.2 Anatomical collection site . 9
6.3 Collection volume . 9
6.4 Collection procedure . 9
6.4.1 General . 9
6.4.2 Obtaining bone marrow by puncturing into the intramedullary canal . 10
6.4.3 Obtaining bone marrow by aspirate . 10
7 Transport of bone marrow samples or hBM-MSCs and associated data to the
biobank .10
8 Reception and traceability of bone marrow or hBM-MSCs and associated data .10
9 Isolation and expansion of hBM-MSCs .11
9.1 Processes . 11
9.2 Unique identification . 11
9.3 Testing for infectious agents . 11
9.4 Isolation of hBM-MSCs from bone marrow samples obtained by puncturing into
the intramedullary canal and primary culture . 11
9.5 Isolation of hBM-MSCs from bone marrow samples obtained by aspirate and
primary culture .12
9.6 Subculture and limited expansion .12
10 Characterization of hBM-MSCs .12
10.1 General .12
10.2 Viability .13
10.3 Morphology . 13
10.4 Population doubling time and subculture/passage. 13
10.4.1 PDT . 13
10.4.2 Subculture/passage . 14
10.5 Cell population purity . 14
10.6 In vitro self-renewal assessment . 14
10.7 Proliferation . 14
10.8 Differentiation capability — In vitro multilineage differentiation .15
10.8.1 General .15
10.8.2 In vitro adipogenic differentiation . 15
10.8.3 In vitro chondrogeneic differentiation . 15
10.8.4 In vitro osteogenic differentiation . 16
10.9 Immunophenotyping by flow cytometry . 16
10.10 Paracrine secretion/expression (protein-based assay of secretome) . 17
10.11 Immunoregulation (modulation of immune cells) . 17
10.12 Microbial contamination . 18
iii
11 Quality control .19
12 Storage .19
13 Thawing .20
14 Disposal .20
15 Distribution of hBM-MSCs — Information for users .21
16 Transport of hBM-MSCs .21
16.1 General . 21
16.2 hBM-MSCs frozen in ampoules or cryovials . 22
16.3 Living hBM-MSC cultures. 22
Annex A (informative) Preparation of human bone marrow mononuclear cells (hBM-MNCs) .23
Bibliography .24
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
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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
Mesenchymal stromal cells are a heterogeneous cell population that is characterized by multiple
functional properties including the ability to secrete paracrine factors, regulate immune effector
[8][9][10][11] [12][13]
cells, maintain primitive phenotypes of other cell populations and support tissue
[14][15]
regeneration. Mesenchymal stromal cells can contain a sub-population of stem or progenitor cells
that demonstrate in vitro self-renewal and differentiation, as has been rigorously demonstrated for
[16]
bone marrow-derived progenitor cells .
[17]
Mesenchymal stromal cells and mesenchymal stem cells are both abbreviated as “MSCs” . For the
purpose of this document, the abbreviated term “MSCs” refers to mesenchymal stromal cells.
The functional definition of MSCs has evolved over time as the biology of these cells is better understood.
Despite these advances, substantial ambiguities persist regarding the nomenclature, nature, identity,
function, mode of isolation and experimental handling of these cells. MSCs are not fully defined by the
[18]
initial minimal criteria, proposed by the International Society of Cell and Gene Therapy (ISCT), and
[19][20]
as such require careful characterization by a matrix of functional assays .
[12][21][22][23][24] [25]
MSCs have been isolated from bone marrow, umbilical cord and other tissue sources,
and are widely used for non-clinical research. MSCs from different tissue sources have different
properties. Different institutions use different practices for isolating, processing and biobanking these
MSCs, 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 MSCs from specific tissue
sources.
This document provides requirements for biobanking of human mesenchymal stromal cells derived
from bone marrow (hBM-MSCs) for research purposes. This document is applicable for academic
centres, public and private institutions performing a biobanking service of hBM-MSCs for research and
development (R&D) and preclinical studies, not for clinical use.
Importantly, this document is focused on MSCs that have been isolated, manipulated and/or propagated
in culture for research purposes.
[26]
ISBT 128 provides terminology and abbreviations for all medicinal products including cell therapy,
and abbreviates these as “MSC(M)” to denote mesenchymal stromal cells from bone marrow. This
document recognizes this abbreviation, but uses the more commonly used convention in research to
[27]
denote human mesenchymal stromal cells derived from bone marrow (hBM-MSCs) .
vi
INTERNATIONAL STANDARD ISO 24651:2022(E)
Biotechnology — Biobanking — Requirements for human
mesenchymal stromal cells derived from bone marrow
1 Scope
This document specifies requirements for the biobanking of human mesenchymal stromal cells derived
from bone marrow (hBM-MSCs), including the collection of bone marrow and associated data, isolation,
culture, characterization, quality control, cryopreservation, storage, thawing, disposal, distribution
and transport.
This document is applicable to all organizations performing biobanking with hBM-MSCs used for
research.
This document does not apply to hBM-MSCs for the purpose of in vivo application in humans, cell
therapy, clinical applications, tissue engineering 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
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO 20387:2018, ISO 21709:2020
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, analysing and distributing defined biological material as well as
related information and data
[SOURCE: ISO 20387:2018, 3.6]
3.4
bone marrow
bone marrow tissue
soft, sponge-like tissue in the centre of most bones which produces white blood cells, red blood cells
and platelets
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 a cell
[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 surface markers, genetic polymorphisms and biological activities
[SOURCE: ISO/TS 22859:2022, 3.8]
3.9
colony forming unit fibroblast
CFU-F
typical in vitro assay to demonstrate self-renewal (3.22) potential of progenitor cells plated at low
frequencies that results in a formation of a colony of fibroblast-looking cells
Note 1 to entry: A count of these colonies is instructive of the colony forming potential or in vitro self-renewal
capacity of these cells.
[SOURCE: ISO/TS 22859:2022, 3.9]
3.10
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]
3.11
differentiation
process to bring the cells into a defined cell state or fate
[SOURCE: ISO/TS 22859:2022, 3.11]
3.12
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]
3.13
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: CLSI H44 -A2: 2004, Clause 4, modified — Note 2 to entry has been added.]
3.14
heterogeneity
non-uniformity of composition, quality or structure of a population of cells
[SOURCE: ISO/TS 22859:2022, 3.14]
3.15
human mesenchymal stromal cell derived from bone marrow
hBM-MSC
heterogeneous cellular population isolated from bone marrow (3.4), which has the ability to modulate
the immune response, secrete paracrine factors, and undergo adipogenesis, osteogenesis and
chondrogenesis in vitro
Note 1 to entry: Without any manipulation, “culture-adapted MSCs” 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.
3.16
licensing
act of stimulating hBM-MSCs (3.15) using inflammatory cytokines to
become more immunosuppressive
Note 1 to entry: Licensing is a biological term and not a regulatory or legal term.
[SOURCE: ISO/TS 22859:2022, 3.17, modified — “hBM-MSCs” has replaced “hUC-MSCs” in the definition.]
3.17
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, modified — “new” added to the definition. Note 1 to entry deleted.]
3.18
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.]
3.19
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.]
3.20
primary culture
culture started from cells, tissues, or organs taken directly from an organism, and before the first
subculture, propagation and consecutive passages (3.17) in vitro
[SOURCE: ISO 21709:2020, 3.16, modified — Note 1 to entry deleted.]
3.21
proliferation
cell number expansion by cell division
3.22
self-renewal
ability of stem cells (3.23) 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 focused functional differentiated cells, while
the other daughter cell still retains the characteristics of the parental stem cell.
[SOURCE: ISO/TS 22859:2022 3.23]
3.23
stem cell
non-specialized cells with the capacity for self-renewal (3.22) and differentiation potential (3.12), 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]
3.24
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]
3.25
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 Abbreviated terms and symbols
ACAN aggrecan
AHR aryl hydrocarbon receptor
ALP alkaline phosphatase
ANGPT2 angiopoietin 2
AP2 adipocyte protein-2
BCL-2 B-cell lymphoma 2
CCL2 chemokine C-C motif ligand 2
CCL7 chemokine C-C motif ligand 7
CCR7 C-C chemokine receptor type 7
CCR10 chemokine receptor type 10
CD clusters of differentiation
CEBPα CCAAT/enhancer-binding protein alpha
CFSE carboxyfluorescein succinimidyl ester
CFU-F colony forming unit fibroblast
CIITA class II major histocompatibility complex trans activator
CO carbon dioxide
COL10 collagen type X
COL2A1 collagen type 2A1
COX-2 cyclooxygenase 2
CX3CR1 CX3C chemokine receptor 1
CXCL9 C-X-C motif chemokine ligand 9
CXCL10 C-X-C motif chemokine ligand 10
CXCL11 C-X-C motif chemokine ligand 11
CXCL12 C-X-C motif chemokine ligand 12
CXCR1 chemokine receptor type 1
CXCR4 chemokine receptor type 4
CXCR6 chemokine receptor type 6
DMEM Dulbecco’s modified eagle medium
EDTA ethylenediaminetetraacetic acid
FBS fetal bovine serum
GAL-1 galectin-1
hBM-MSCs human mesenchymal stromal cells derived from bone marrow
HBV hepatitis B virus
HCV hepatitis C virus
HGF hepatocyte growth factor
HIV human immunodeficiency virus
HLA human leukocyte antigen
HLA-DR human leukocyte antigen DR
HO-1 heme oxygenase-1
HSP70A heat shock protein 1
HSP70B heat shock protein 70B
ICAM-1 intercellular adhesion molecule 1
IDO indoleamine 2,3-dioxygenase 1
IFN-γ interferon-gamma
IL-1RA interleukin-1 receptor antagonist
IL-6 interleukin-6
KGF keratinocyte growth factor
LPL lipoprotein lipase
MSCs mesenchymal stromal cells
MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
N count of cells harvested
N count of cells seeded
OCN osteocalcin
OPN osteopontin
P starting population of the cells
PBS phosphate-buffered saline
PCR polymerase chain reaction
PDL-1 programmed death-ligand 1
PDT population doubling time
PPAR-γ peroxisome proliferator-activated receptor gamma
QC quality control
R&D research and development
RUNX2 Runt-related transcription factor 2
SOX9 SRY-related HMG box 9
T end time point of incubation, in hours
T starting time point of incubation, in hours
TGF-β transforming growth factor beta
TIMP-1 tissue inhibitor of metalloproteinases 1
TIMP-2 tissue inhibitor of metalloproteinases 2
TLR-4 toll-like receptor 4
TP treponema pallidum
TSG-6 tumour necrosis factor-inducible gene 6 protein
ULBP-3 UL16 binding protein 3
VCAM-1 vascular cell adhesion molecule 1
VEGF vascular endothelial growth factor
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 isolation, culture, storage, thawing and
transport of hBM-MSCs.
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
hBM-MSCs 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 hBM-MSCs, procedures, QC documents for collection, separation,
expansion, storage, transportation and testing, and data analysis shall be established, documented,
implemented, regularly reviewed and updated.
The authenticity and properties of hBM-MSCs shall be monitored throughout the complete biobanking
process from isolation to distribution.
5.2 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 hBM-MSC generation,
characterization, culture, cryopreservation, thawing and transport.
hBM-MSC services provided by external operators shall demonstrate relevant professional knowledge,
experience and corresponding skills, and regularly conduct documented personnel training and
assessment.
The biobank shall ensure that facilities and environmental conditions do not adversely affect hBM-MSC
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 hBM-MSCs and the need for aseptic processing.
5.3 Reagents, consumables and other supplies
ISO 21709:2020, 4.5, shall be followed.
The biobank shall establish acceptance criteria for materials, including reagents and consumables,
necessary for hBM-MSC isolation, culture, storage, thawing and transport.
5.4 Management of information and data
ISO 20387:2018, 7.8.3 and 7.10, shall be followed.
The biobank shall manage and maintain related data of hBM-MSCs, including but not limited to the
following:
a) the technical information: methods used in the derivation of cells, culture conditions, passage data
including the passage number, characterization and microbiological test data;
b) the preservation and storage information;
c) the safety testing data.
Certain data retention times, data integrity and security of data storage shall be ensured.
For hBM-MSCs, 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 bone marrow samples and associated data
6.1 Information about the bone marrow donor
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 bone marrow donor (e.g. statement of donor health or suitability,
disease type, concomitant disease, demographics such as age and sex);
c) the information about medical treatment and special treatment prior to bone marrow collection
(e.g. date, terms of treatment, medication, conclusion of medical specialist);
d) the negative test result for hepatitis B and C, HIV, TP and toxoplasmosis unless a positive test result
is needed for a specific research purpose;
NOTE 1 Additional virus testing can be considered where relevant.
e) 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 donor’s 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.
Unless related to a specific research purpose, donors shall not be considered suitable for donation, if
they:
— were unhealthy at the time of donation;
NOTE 2 Deferral periods for specific infections can exist and/or vary in accordance with local regulatory
guidelines for other cell and tissue products.
— have tested positive for at least one infectious disease, see 6.1 d).
During the collection process for human cells, measures shall be taken to protect donor and biobank
personnel health and safety.
The isolation of hBM-MSCs should not be performed from donors with medical contraindications for
extraction of bone marrow aspirate, sedation or anaesthesia based on a related risk assessment.
It has been shown that the status of bone marrow donors such as age, viral infections, neoplastic and
[28]
immunological disorders affect some of the characteristics of MSCs .
6.2 Anatomical collection site
With regard to the anatomical collection site, recommendations are given by the International Council
[29]
for Standardization in Hematology (ICSH) protocols .
If the bone marrow is collected by puncturing into the intramedullary canal, the collection can be made
[12][21][30]
from the head of the femur .
The anatomical collection site shall be documented for each procedure.
6.3 Collection volume
It has been shown that the volume of bone marrow collection influences the number of isolated
mononuclear cells, and therefore plays an important role in the efficient isolation of hBM-MSCs from
[21]
bone marrow. Thus:
a) for bone marrow aspirates, a minimum volume of 10 ml should be collected and shall be
[24][31][32][33]
documented ;
b) for samples obtained by puncturing into the intramedullary canal, a minimum volume of 15 ml
[12][13][21][30]
should be collected and shall be documented .
6.4 Collection procedure
6.4.1 General
ISO 20387:2018, 7.2, shall be followed.
The biobank shall establish, implement, validate and document a procedure for the collection of bone
marrow for each used collection method (see 6.4.2 and 6.4.3).
All reagents and materials used to collect the bone marrow shall be sterile.
[34]
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.
6.4.2 Obtaining bone marrow by puncturing into the intramedullary canal
The sample should be collected into a disposable sterile container, and filled with an anticoagulant.
Internationally recognized and validated anticoagulants should be EDTA (ethylenediaminetetraacetic
acid) or heparin at 15 IU/ml to 25 IU/ml.
6.4.3 Obtaining bone marrow by aspirate
Bone marrow aspiration can be performed in accordance with the International Council for
[29]
Standardization in Hematology (ICSH) protocols .
[29]
Bone marrow aspirates should be taken in EDTA or heparinized syringes of 15 IU/ml to 25 IU/ml
.
7 Transport of bone marrow samples or hBM-MSCs and associated data to the
biobank
ISO 20387:2018, 7.4, shall be followed. ISO/TS 20658 can be used to consider transport, handling and
safety requirements for facilities.
[34]
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 bone marrow from the
collection facility to the biobank. Instructions on the transportation of bone marrow to the preparation
site as well as the transportation of hBM-MSC preparations to the biobank should be included.
The following factors shall be taken into account for transportation of bone marrow:
a) packaging, material, containers and secondary containment;
b) medium or solvent;
c) transportation duration, temperature and temperature monitors.
Biological source material collection medium 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 bone marrow or hBM-MSCs and associated data
ISO 20387:2018, 7.3.1, 7.3.2, 7.5, shall be followed.
9 Isolation and expansion of hBM-MSCs
9.1 Processes
For establishing hBM-MSCs, ISO 21709:2020, 5.1, shall be followed.
The biobank shall establish, implement, validate, document and maintain procedures for hBM-MSC
isolation in primary culture and subculture depending on the method used for collection (see 6.4.2 and
6.4.3).
Processes should be performed in a biosafety cabinet or under a laminar flow hood using appropriate
aseptic techniques.
9.2 Unique identification
The unique identification of hBM-MSCs 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.
9.3 Testing for infectious agents
The cells derived from the donor biological material should be tested for relevant transmittable
infectious agents, e.g. HIV, HBV, HCV, 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 established cells.
9.4 Isolation of hBM-MSCs from bone marrow samples obtained by puncturing into the
intramedullary canal and primary culture
For optimal functionality, the hBM-MSC isolation procedure should be started within 2 h but not beyond
[21][35]
8 h after the bone marrow has been collected (see 6.4.2) .
Procedures for the isolation of hBM-MSCs from bone marrow can vary. The following workflow should
be followed:
a) Perform a sterile mechanical disruption of puncturing into the intramedullary canal to create a cell
suspension. The cell suspension can be made as described in Annex A, b).
b) Filter the resulting sample, e.g. see Annex A, c).
c) Collect the filtered sample and centrifuge it, e.g. see Annex A, d) and e).
d) Collect cells (buffy coat) and dilute with PBS. Antibiotics should be added. Subsequently,
mononuclear cells need to be separated by density gradient centrifugation. See Annex A, f) to j) for
exemplary workflow steps.
e) Once the mononuclear cells have been separated, perform a cell count and determine cell viability
using a dye exclusion technique (e.g. trypan blue) either manually or using an automated device.
f) It is suggested to initiate the cultures when cell viability is greater than 90 % and the cell count is
3 2 3 2
between 100 × 10 cells/cm to 160 × 10 cells/cm . Culture conditions should be documented, e.g.
O tension, pH, temperature, humidity conditions and normoxic hypoxic conditions.
g) After 72 h of culture, remove non-adherent cells and add fresh culture medium. Do not remove the
culture medium to observe adherent cells (approximately 5 days to 7 days).
h) When a confluency of adherent cells is observed between 70 % to 80 %, passage the cells by
trypsinization. Serum free media should be used for culture. Alternatively, the use of culture
medium supplemented with FBS (typically 5 % to 10 %), platelet lysate (typically 5 % to 10 %) or
human serum (typically 5 % to 10 %) is also suggested.
9.5 Isolation of hBM-MSCs from bone marrow samples obtained by aspirate and
primary culture
In order to maintain the cell viability of the hBM-MSCs, the hBM-MSC isolation procedure shall be
performed within a pre-determined time limit.
a) Transfer the bone marrow aspirate into (a) sterile tube(s) and centrifuge them. Then resuspend the
[35][36][37].
cells in a culture medium An exemplary workflow can be found in Annex A.
b) Perform a cell count and determine the viability of the cells.
c) Direct seeding: the culture should be started with a cell viability ≥ 90 % and a cell density of
6 2
1 × 10 cells/cm .
d) Incubate at 36,5 °C ± 0,5 °C, with 5 % CO and 95 % relative humidity. Then, follow 9.4, f) and g).
e) hBM-MSCs obtained from bone marrow aspirate should be grown after the first passage, at a
3 2 3 2
density of 3 × 10 cells/cm to 7 × 10 cells/cm .
It is suggested not to use the hBM-MSCs after the fifth passage, because the probability of phenotypic
[38][39][40]
and chromosomal instability increases .
9.6 Subculture and limited expansion
A culture can be further expanded for biobanking after successful establishment of the primary
culture; 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, yeast,
mycoplasma, endotoxins and adventitious viral agents) before any further expansion.
Cell passaging follows the relative protocols after establishment of primary culture. Expansion of
hBM-MSCs is recommended for up to three 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. CFU-F, undifferentiation status and immunophenotyping).
10 Characterization of hBM-MSCs
10.1 General
The biobank shall establish, document and implement procedures to characterize hBM-MSCs and report
the relevant data so that users can determine suitability for their intended use.
The biobank shall establish a matrix of assays and a set of markers based at least on Clause 10.
The biobank shall perform ongoing characterization of hBM-MSCs in culture. These characterizations
shall include but are not limited to:
a) authentication;
b) cell morphology;
c) growth kinetics: can be calculated using PDT;
d) viability;
e) differentiation capability in vitro;
f) immunophenotype;
g) functional characterization in vitro;
h) being free of microbial contamination.
10.2 Viability
The biobank shall establish, 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 of the cell culture at regular intervals and especially
after changes of cell culture conditio
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