ISO/TS 22859:2022

TECHNICAL ISO/TS
SPECIFICATION 22859
First edition
2022-07
Biotechnology — Biobanking
— Requirements for human
mesenchymal stromal cells derived
from umbilical cord tissue
Biotechnologie — Banques biologiques — Exigences relatives aux
cellules stromales mésenchymateuses humaines issues des tissus du
cordon ombilical
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 .10
5.1 General . 10
5.2 Personnel, facilities and equipment . 10
5.3 Reagents, consumables and other supplies . 11
5.4 Management of information and data . 11
6 Collection of umbilical cord and associated data .11
6.1 Information about the umbilical cord donor . 11
6.2 Collection procedure .12
7 Transport of umbilical cord or hUC-MSCs and associated data to the biobank .13
8 Reception and traceability of umbilical cord tissue or hUC-MSCs and associated data .13
9 Isolation and expansion of hUC-MSCs .13
9.1 Processes .13
9.2 Unique identification . 13
9.3 Testing for infectious agents . 14
9.4 Isolation of hUC-MSCs and primary culture . 14
9.5 Subculture and limited expansion . 14
10 Characterization of hUC-MSCs .14
10.1 General . 14
10.2 Viability . 15
10.3 Morphology . 15
10.4 Population doubling time and subculture/passage. 16
10.4.1 PDT . 16
10.4.2 Subculture/passage . 16
10.5 Cell population purity . 16
10.6 In vitro self-renewal assessment . 17
10.7 Proliferation . 17
10.8 Differentiation capability — In vitro multilineage differentiation . 17
10.8.1 General . 17
10.8.2 In vitro adipogenic differentiation . 17
10.8.3 In vitro chondrogenic differentiation . 18
10.8.4 In vitro osteogenic differentiation . 18
10.9 Immunophenotyping by flow cytometry . 18
10.10 Paracrine secretion/expression (protein-based assay of secretome) .20
10.11 Immunoregulation (modulation of immune cells) . 20
10.12 Microbial contamination . 21
11 Quality control .21
12 Storage .22
13 Thawing .23
14 Disposal .24
15 Distribution of hUC-MSCs — Information for users .24
16 Transport of hUC-MSCs .24
iii
16.1 General . 24
16.2 hUC-MSCs frozen in ampoules or cryovials . 25
16.3 Living hUC-MSC cultures . 25
Annex A (informative) Exemplary quality control test procedure for biobanking of hUC-
MSCs .27
Annex B (informative) Examples for suitable methods for the isolation and primary culture
of hUC-MSCs .28
Annex C (informative) Exemplary methods for characterization of hUC-MSCs .30
Bibliography .32
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
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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
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Attention is drawn to the possibility that some of the elements of this document may be the subject of
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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
[8]
functional properties including the ability to secrete paracrine factors, regulate immune effector
[9][10] [11][12]
cells, maintain primitive phenotypes of other cell populations and support tissue
[13][14]
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
[15]
umbilical cord-derived progenitor cells.
[16]
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
[17]
initial minimal criteria proposed by the International Society of Cell and Gene Therapy (ISCT), and
[16]
as such require careful characterization by a matrix of functional assays.
[15] [18][19]
MSCs have been isolated from umbilical cord , bone marrow 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 umbilical cord tissue (Wharton’s jelly) (hUC-MSCs) for research purposes. This document is
applicable for academic centres, public and private institutions performing a biobanking service of
hUC-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
from umbilical cord tissue (also called “Wharton’s jelly”) in culture for research purposes. These cells
are different from unmanipulated cells found in human umbilical cord tissue (Wharton’s jelly).
[20]
ISBT 128 provides terminology and abbreviations for all medicinal products including cell therapy,
and abbreviates these as “MSC(WJ)” to denote mesenchymal stromal cells from Wharton’s jelly. This
document recognizes this abbreviation, but uses the more commonly-used convention in research to
denote human mesenchymal stromal cells derived from umbilical cord tissue (Warton’s jelly) (hUC-
[21]
MSCs).
vi
TECHNICAL SPECIFICATION ISO/TS 22859:2022(E)
Biotechnology — Biobanking — Requirements for human
mesenchymal stromal cells derived from umbilical cord
tissue
1 Scope
This document specifies requirements for the biobanking of human mesenchymal stromal cells
derived from umbilical cord tissue (i.e. Wharton’s jelly), further referred to as hUC-MSCs, including
the collection of umbilical cord tissue and associated data, isolation, culture characterization, quality
control, cryopreservation, storage, thawing, disposal, distribution and transport.
This document is applicable to all organizations performing biobanking of hUC-MSCs used for research
and development.
This document does not apply to hUC-MSCs 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
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
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
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
3.6
cell master file
complete dossier of all procedures and records used to generate a cell
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
3.9
colony forming unit fibroblast
CFU-F
typical in vitro assay to demonstrate self-renewal (3.23) 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.
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
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
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
3.15
homogeneity
uniformity of composition, quality or structure of a population of cells
3.16
human mesenchymal stromal cell derived from umbilical cord tissue
hUC-MSC
heterogeneous cellular population isolated from umbilical cord (3.25), 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.
3.17
licensing
act of stimulating hUC-MSCs (3.16) using inflammatory cytokines to
become more immunosuppressive
Note 1 to entry: Licensing is a biological term and not a regulatory or legal term.
3.18
passage
subculture
process of further culturing of cells in a culture vessel to provide higher surface area/volume for the
cells to grow
Note 1 to entry: A passage can be performed by harvesting an aliquot from the parent vessel and reseeding it into
another vessel.
3.19
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.]
3.20
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.]
3.21
primary culture
culture started from cells, tissues, or organs taken directly from an organism, and before the first
subculture, propagation and consecutive passages (3.18) in vitro
[SOURCE: ISO 21709:2020, 3.16, modified — Note 1 to entry deleted.]
3.22
proliferation
cell number expansion by cell division
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 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.
3.24
stem cell
non-specialized cells with the capacity for self-renewal (3.23) 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.
3.25
umbilical cord
umbilical cord tissue
UC
soft, gelatinous connective tissue (i.e. Wharton jelly), excluding umbilical arteries, umbilical vein and
placenta
3.26
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]
3.27
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
2D1 clone of anti-human CD45 antibody
4F2 clone of anti-human CD98 antibody
561 clone of anti-human CD34 antibody
581 clone of anti-human CD34 antibody
58XB4 clone of anti-human CD104 antibody
63D3 clone of anti-human CD14 antibody
A20 tumour necrosis factor alpha-induced protein 3 (TNFAIP3)
ACAN aggrecan
AD2 anti-human CD-73 (Ecto-5’-nucleotidase) antibody
AHR aryl hrdrocarbon receptor
ALP alkaline phosphatase
ANGPT2 angiopoietin-2
AP2 adipocyte protein-2
α-SMA alpha-smooth muscle actin
B7RP2 B7-related protein 2
BCL-2 B-cell lymphoma 2
BJ18 clone of anti-human CD44 antibody
BM bone marrow
C44Mab-5 clone of anti-human CD44 antibody
CCL2 C-C Motif Chemokine ligand 2
CCL5 C-C Motif Chemokine ligand 5
CCL7 C-C Motif Chemokine ligand 7
CCR7 C-C Motif Chemokine receptor 7
CCR10 C-C Motif Chemokine receptor 10
CD clusters of differentiation
CD9 clusters of differentiation 9
CD13 clusters of differentiation 13
CD14 clusters of differentiation 14
CD29 clusters of differentiation 29
CD31 clusters of differentiation 31
CD34 clusters of differentiation 34
CD44 clusters of differentiation 44
CD45 clusters of differentiation 45
CD46 clusters of differentiation 46
CD55 clusters of differentiation 55
CD73 clusters of differentiation73
CD90 clusters of differentiation 90
CD98 clusters of differentiation 98
CD104b clusters of differentiation 104b
CD105 clusters of differentiation 105
CD 146 clusters of differentiation146
CD276 clusters of differentiation 276
CEBPα enhancer-binding protein alpha
CFSE carboxyfluorescein succinimidyl ester
CFU colony forming units
human gene which encodes a protein called the class 2, major histocompatibility
CIITA
complex, transactivator
CO carbondioxide
COL2A1 collagen type 2 alpha 1
COX-2 prostaglandin-endoperoxide synthase 2 (cyclooxygenase-2)
CX3CR1 C-3X-C Motif 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 C-X-C Motif Chemokine receptor type 1
CXCR4 C-X-C Motif Chemokine receptor type 4
CXCR6 C-X-C Motif Chemokine receptor type 6
DCN.70 anti-CD276 (B7-H3) antibody
DMEM Dulbecco’s modified eagle medium
DMEM-LG Dulbecco’s modified eagle medium low glucose
DMSO dimethyl sulfoxide
DRAP-24 clone of anti-human CD9 antibody
EDTA ethylenediaminetetraacetic acid
ELISA enzyme-linked immunosorbent assay
FABP4 Fatty acid-binding protein 4
FACS fluorescence activating cell sorter
FBS fetal bovine serum
FRP-1 frizzled-related protein
GAL-1 galactose-1
HBsAg hepatitis B surface antigen
HBV hepatitis B virus
HCD14 clone of anti-human CD14 antibody
HCsAg hepatitis C surface antigen
HCV hepatitis C virus
HEL113 gene of vimentin
HGF hepatocyte growth factor
HI30 clone of anti-human CD45 antibody
HI9a clone of anti-human CD9 antibody
HIV human immunodeficiency virus
HLA human leukocyte antigen
HLA-Class I human leukocyte antigen – Class I
HLA-Class II human leukocyte antigen – Class II
HLA-DR human leukocyte antigen DR
HO-1 haem oxygenase-1
HSP70A heat shock protein 70A
HSP70B heat shock protein 70B
hUC-MSC human umbilical cord mesenchymal stromal cell
ICAM-1 intercellular adhesion molecule-1
IDO indoleamine 2,3-dioxygenase 1
IFN-γ interferon-gamma
IFU instructions for use
IL-1 interleukin-1
IL-1RA interleukin-1 receptor antagonist
IL-6 interleukin-6
IL-8 interleukin-8
INCAM-110 inducible cell adhesion molecule 110
ITGB1 integrin 1 antibody
ITGB4 integrin 4 antibody
KGF keratinocyte growth factor
L243 clone of anti-human HLA-DR antibody
LN3 clone of anti-HLA-DR antibody
LPL lipoprotein lipase
MCAM melanoma cell adhesion molecule
MEM minimum essential media
MEM-108 minimum essential media-108
MIH42 molting inhibits hormones-42
MPR-1 mannose 6-phosphate receptor-1
MR106 clone of anti-rat CD106 antibody
mRNA messenger RNA
MSCs mesenchymal stromal cells
My10 anti-CD34 antibody
N count of cells harvested
N count of cells seeded
O91D3 clone of anti-vimentin antibody
O92E4 clone of anti-human CD31 antibody
OPN osteopontin
P starting culture passage
P1H12 anti-CD146 antibody
PBS phosphate-buffered saline
PCR polymerase chain reaction
PDL-1 programmed death-ligand 1
PDL-2 programmed death-ligand 2
PDT population doubling time
PECAM-1 platelet endothelial cell adhesion molecule-1
PI9 blast resistance gene PI 9
Poly29291 poly sulfuryl dichloride
PPAR-γ peroxisome proliferators-activated receptor-gamma
PREF-1 preadipocytokine 1
QA17A19 clone of anti-human CD45 recombinant antibody
QC quality control
SHM-57 anti-human CD146 (MUC18, Mel-cam) antibody
SOX2 SRY-related HMG box 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-β
Thy-1 thymus antigen
TIMP-1 tissue inhibitor of metalloproteinases 1
TIMP-2 tissue inhibitor of metalloproteinases 2
TLR3 toll-like receptor-3
TLR4 toll-like receptor-4
TNF tumour necrosis factor
TP Treponema pallidum
TRAIL tumour necrosis factor-related apoptosis-inducing ligand
TS2/16 clone of anti-human CD29 antibody
TSE transmissible spongiform encephalopathies
TSG-6 tumour necrosis factor-inducible gene 6 protein
TSP-1180 thrombin-sensitive protein-1180
UC umbilical cord
UCHL1 ubiquitin carboxy-terminal hydrolase L1
ULBP-3 UL16 binding protein 3
VCAM-1 vascular cell adhesion molecule-1
VEGF vascular endothelial growth factor
W16220A clone of anti-vimentin antibody
W6/32 clone of anti-HLA class I antibody
WM15 CD13 antibody
WM59 CD31 antibody
WNTS Wnt family
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 hUC-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
hUC-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 hUC-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 donor’s health status and characteristics, tissue harvest conditions as well as culture methods
can influence the properties of hUC-MSCs. Workflow steps cannot always be controlled. Thus, their
impact on the hUC-MSCs properties for biobanking shall be investigated, and mitigation measures shall
be established to enable the required quality control. In these cases, quality risk management (QRM)
should be taken into account.
The biobank shall assess biorisks of umbilical cord and hUC-MSCs at the facilities and implement
appropriate biosafety measures for the protection of personnel and environment.
The authenticity and properties of hUC-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 hUC-MSC generation,
characterization, culture, cryopreservation, thawing and transport.
The biobank shall ensure that external operators providing hUC-MSC services demonstrate relevant
knowledge, experience and corresponding skill.
The biobank shall ensure that facilities and environmental conditions do not adversely affect hUC-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 hUC-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 hUC-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 associated data of hUC-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 hUC-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 umbilical cord and associated data
6.1 Information about the umbilical cord 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 umbilical cord donor (e.g. statement of donor health or suitability,
disease type, concomitant disease, demographics such as gestational age and age);
c) the information about medical treatment and special treatment prior to umbilical cord collection
(e.g. date, terms of treatment, medication, conclusion of medical specialist);
d) the negative test result for hepatitis B and C, HIV and TP 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;
f) the absence of congenital abnormalities in the neonates, when appropriate.
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.
6.2 Collection procedure
ISO 20387:2018, 7.2, shall be followed.
The biobank shall establish, implement, validate and document a procedure for the collection of
umbilical cord.
Umbilical cord tissue collection should be operated under the aseptic conditions to avoid sample
contamination.
All reagents and materials used to collect the umbilical cord tissue shall be sterile.
[22]
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.
Umbilical cord tissue can be collected following vaginal or caesarean delivery. It shall be ensured that
collection procedures do not interfere with standard delivery practices or the safety of a newborn or
the mother. The collection procedure shall take place at the distal end of the cord.
The required amount of cord should be determined in consideration of the intended purpose of the
biobank or user. Both ends of the cord shall be clamped to prevent contamination.
NOTE 1 Length of the cord is discussed in Reference [23].
NOTE 2 Mass can also be used to determine required amount of cord.
NOTE 3 The following biological material can be considered for testing data:
a) cord blood (for detection of certain antigens and/or antibodies against such antigen);
b) cultured mesenchymal stromal cells from umbilical cord (for PCR testing to detect expression of certain
gene(s));
c) mother’s blood (for detection of certain antigens and/or antibodies against such antigen).
Blood should not be drawn from a newborn.
In case of vaginal delivery, a procedure to minimize bacterial and fungal contamination of collected
umbilical cord shall be established, implemented and documented. Such a procedure can include rinsing
the collected umbilical cord and content with a rinsing solution, e.g. sterile phosphate-buffered saline
solution (1⨯PBS) containing heparin and/or penicillin, streptomycin and/or Amphotericin B.
The medium for umbilical cord collection should be established in consideration of the intended
purpose of the biobank and/or user.
A recommended medium for umbilical cord collection is sterile phosphate-buffered saline solution
(1⨯PBS) containing heparin and/or penicillin, streptomycin and/or Amphotericin B.
7 Transport of umbilical cord or hUC-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.
[22]
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 umbilical cord
from the collection facility to the biobank. Instructions on the transportation of umbilical cord to
the preparation site as well as the transportation of hUC-MSC preparations to the biobank should be
included.
[24]
EXAMPLE The temperature can be between 2 °C and 8 °C .
The following factors shall be taken into account for transportation of umbilical cord tissue:
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 umbilical cord tissue or hUC-MSCs and
associated data
ISO 20387:2018, 7.3.1, 7.3.2 and 7.5, shall be followed.
9 Isolation and expansion of hUC-MSCs
9.1 Processes
For establishing hUC-MSCs, ISO 21709:2020, 5.1, shall be followed.
The biobank shall establish, implement, validate, document and maintain procedures for hUC-MSC
isolation in primary culture and subculture.
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 hUC-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 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 hUC-MSCs and primary culture
There are various methods of isolation and establishing a primary culture including explant or
enzymatic methods (see Annex B for exemplary methods).
The biobank shall document and provide information about the section of the cord used for the isolation
of the cells (e.g. perivascular region, Wharton’s jelly).
NOTE Current methods to isolate hUC-MSCs from human umbilical cord (hUC) yield low amounts of cells
with variable proliferation potentials. Although hUC is an anatomically-complex organ, differences in MSC
[25][26]
properties can appear due to differences in the anatomical regions within the Wharton’s jelly .
It is recommended to mechanically separate the blood vessels from the Wharton’s jelly before isolating
hUC-MSCs. Umbilical cord tissue should be fragmented small enough (1 mm to 3 mm) to allow the cells
to come into contact with gases and nutrients.
Several different culture media have been used to isolate hUC-MSCs such as MEM, DMEM-LG. The
biobank shall document and provide the composition or formula of the medium to the hUC-MSCs
recipient/user.
9.5 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 bacter
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