Methods in molecular biology vol 1590 stem cell banking concepts and protocols

Methods in
Molecular Biology 1590

Jeremy M. Crook
Tenneille E. Ludwig Editors

Stem Cell
Banking
Concepts and Protocols


METHODS

IN

MOLECULAR BIOLOGY

Series Editor
John M. Walker
School of Life and Medical Sciences
University of Hertfordshire
Hatfield, Hertfordshire, AL10 9AB, UK

For further volumes:
/>

Stem Cell Banking
Concepts and Protocols

Edited by

Jeremy M. Crook
ARC Centre of Excellence for Electromaterials Science, Intelligent Polymer Research Institute, AIIM Facility,
Innovation Campus, University of Wollongong, Fairy Meadow, NSW, Australia
Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, NSW, Australia
Department of Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia

Tenneille E. Ludwig
WiCell Research Institute, Madison, WI, USA


Editors
Jeremy M. Crook
ARC Centre of Excellence for Electromaterials
Science
Intelligent Polymer Research Institute
AIIM Facility, Innovation Campus
University of Wollongong
Fairy Meadow, NSW, Australia

Tenneille E. Ludwig
WiCell Research Institute
Madison, WI, USA

Illawarra Health and Medical Research Institute
University of Wollongong
Wollongong, NSW, Australia
Department of Surgery
St Vincent’s Hospital
The University of Melbourne
Fitzroy, VIC, Australia

ISSN 1064-3745
ISSN 1940-6029 (electronic)
Methods in Molecular Biology
ISBN 978-1-4939-6919-7
ISBN 978-1-4939-6921-0 (eBook)
DOI 10.1007/978-1-4939-6921-0
Library of Congress Control Number: 2017934046
© Springer Science+Business Media LLC 2017
This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is
concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction
on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation,
computer software, or by similar or dissimilar methodology now known or hereafter developed.
The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not
imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and
regulations and therefore free for general use.
The publisher, the authors and the editors are safe to assume that the advice and information in this book are believed to
be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty,
express or implied, with respect to the material contained herein or for any errors or omissions that may have been made.
The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Printed on acid-free paper
This Humana Press imprint is published by Springer Nature
The registered company is Springer Science+Business Media LLC
The registered company address is: 233 Spring Street, New York, NY 10013, U.S.A.


Preface
Stem cell banking has a critical role to play for supporting high quality research and transcending the clinical potential of stem cells to actual medicine. Ideally, this is achieved by operating
within a regulatory framework of good laboratory practice (GLP) or good manufacturing
practice (GMP) for standardized, optimized, and controlled cell line production, storage, and

distribution. Among other benefits, creating repositories of quality “seed stock” is a most
immediate way to circumvent the problems associated with extended cell culture, including
susceptibility to genetic and phenotypic drift during propagation, loss of cells due to crosscontamination with microorganisms or other cell lines, and stem cell differentiation.
In recognizing the need for modern banking systems, major developed nations including the US, UK, and Japan have invested significantly in stem cell banking to prepare for
the next major phase in researching and commercializing stem cells and producing clinical
treatments.
Importantly, stem cell banking need not entail setting up large and expensive standalone facilities that operate on a national or international scale, but can involve smaller initiatives to support the activities of individual universities, research institutes, or laboratories.
Whatever the scale, a bank should align with global “best practice” for handling stem cells,
ideally endorsed by leading stem cell organizations, networks, and consortia around the
world. Moreover, a bank should ensure the management and distribution of cell lines in the
most efficient and cost-effective way. For example, the succession of commercial and clinical
aspirations could be facilitated by having low-cost quality-controlled GLP cells for research
that are also available as more expensive clinical-grade GMP lines. In addition, research and
clinical-grade variants of the same cell lines/banks will provide consistency between laboratory and clinical activities for more predictable and better translational application.
Given the recent upsurge in stem cell research and development (R&D), including
technological breakthroughs in creating new types of stem cells such as induced pluripotent
stem cells (iPSCs), as well as clinical trials of human stem cell-based therapies, the publication of this book on Stem Cell Banking is timely.
This volume brings together contributions from experts in the field to guide stem cell
banking, and in turn champion quality stem cell R&D and facilitate the translation of stem
cells to clinical practice. The book covers concepts and protocols relating to the banking of
both pluripotent and somatic stem cells, from the ethical procurement of tissues and cells
for the provision of “seed stock,” standardized methods for deriving hESCs and iPSCs,
isolating mesenchymal stem cells, cell culture and cryopreservation, in addition to quality
assurance (including cell line characterization) and information management.
As a volume in the highly successful Methods in Molecular Biology™ series, it aims to
contribute to the development of competence in the subject by providing advice that is crucial
to establishing a bona fide stem cell bank. By proffering Stem Cell Banking, we hope to
strengthen and maximize the use of existing and future stem cell resources. Finally, the volume
should serve as a valuable resource for established stem cell scientists and those new to the field.
Wollongong, NSW, Australia

Jeremy M. Crook

v


Contents
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

PART I

GENERIC THEMES IN STEM CELL BANKING

1 Stem Cell Banking: A Global View . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Glyn Stacey
2 Quality Assurance in Stem Cell Banking: Emphasis on Embryonic
and Induced Pluripotent Stem Cell Banking . . . . . . . . . . . . . . . . . . . . . . . . . .
Therése Kallur, Pontus Blomberg, Sonya Stenfelt, Kristian Tryggvason,
and Outi Hovatta
3 Acquisition and Reception of Primary Tissues, Cells,
or Other Biological Specimens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Lyn E. Healy
4 Information Management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Alberto Labarga, Izaskun Beloqui, and Angel G. Martin
5 Cryopreservation: Vitrification and Controlled Rate Cooling. . . . . . . . . . . . . .
Charles J. Hunt
6 Quality Assured Characterization of Stem Cells for Safety in Banking
for Clinical Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Kevin W. Bruce, John D.M. Campbell, and Paul De Sousa

7 Ethics and Governance of Stem Cell Banks . . . . . . . . . . . . . . . . . . . . . . . . . . .
Donald Chalmers, Peter Rathjen, Joy Rathjen, and Dianne Nicol

PART II

v
ix

3

11

17
29
41

79
99

PROTOCOLS FOR PLURIPOTENT STEM CELL BANKING

8 Derivation of Human Embryonic Stem Cells. . . . . . . . . . . . . . . . . . . . . . . . . .
Jeremy M. Crook, Lucy Kravets, Teija Peura, and Meri T. Firpo
9 Derivation of Human-Induced Pluripotent Stem Cells in Chemically
Defined Medium. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Guokai Chen and Mahendra Rao
10 Culture, Adaptation, and Expansion of Pluripotent Stem Cells . . . . . . . . . . . .
Jennifer L. Brehm and Tenneille E. Ludwig
11 Cryobanking Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Jeremy M. Crook, Eva Tomaskovic-Crook, and Tenneille E. Ludwig

12 Genome Editing in Human Pluripotent Stem Cells . . . . . . . . . . . . . . . . . . . . .
Jared Carlson-Stevermer and Krishanu Saha

vii

115

131
139
151
165


viii

Contents

PART III

PROTOCOLS FOR MESENCHYMAL STEM CELL BANKING

13 Isolation, Culture, and Expansion of Mesenchymal Stem Cells . . . . . . . . . . . .
Izaskun Ferrin, Izaskun Beloqui, Lorea Zabaleta, Juan M. Salcedo,
Cesar Trigueros, and Angel G. Martin
14 Cryobanking Mesenchymal Stem Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Andrés Pavón, Izaskun Beloqui, Juan M. Salcedo, and Angel G. Martin

PART IV

177

191

PROTOCOLS FOR HUMAN NEURAL STEM CELL BANKING

15 Culturing and Cryobanking Human Neural Stem Cells . . . . . . . . . . . . . . . . . .
Jeremy M. Crook and Eva Tomaskovic-Crook

199

Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

207


Contributors
IZASKUN BELOQUI • StemTek Therapeutics, Derio, Spain
PONTUS BLOMBERG • Vecura, Karolinska University Hospital, Stockholm, Sweden
JENNIFER L. BREHM • WiCell Research Institute, Madison, WI, USA
KEVIN W. BRUCE • Censo Biotechnologies Ltd and Roslin Cell Sciences Ltd, Midlothian, UK
JOHN D.M. CAMPBELL • Scottish Blood Transfusion Service, Edinburgh, UK
JARED CARLSON-STEVERMER • Department of Biomedical Engineering, University of
Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery, University
of Wisconsin-Madison, Madison, WI, USA
DONALD CHALMERS • Centre for Law and Genetics, Faculty of Law, University of
Tasmania, Hobart, TAS, Australia
GUOKAI CHEN • Faculty of Health Sciences, University of Macau, Taipa, Macau, China;
Center for Molecular Medicine, National Heart, Lung and Blood Institute, Bethesda,
MD, USA
JEREMY M. CROOK • ARC Centre of Excellence for Electromaterials Science, Intelligent

Polymer Research Institute, AIIM Facility, Innovation Campus, University of
Wollongong, Fairy Meadow, NSW, Australia; Illawarra Health and Medical Research
Institute, University of Wollongong, Wollongong, NSW, Australia; Department of
Surgery, St Vincent’s Hospital, The University of Melbourne, Fitzroy, VIC, Australia
IZASKUN FERRIN • StemTek Therapeutics, Derio, Spain
MERI T. FIRPO • Division of Endocrinology and Stem Cell Institute, Department
of Medicine, McGuire Translational Research Facility, University of Minnesota,
Minneapolis, MN, USA
LYN E. HEALY • The Francis Crick Institute, London, UK
OUTI HOVATTA • CLINTEC, Karolinska Institute, Flemingsberg, Sweden
CHARLES J. HUNT • UK Stem Cell Bank, National Institute for Biological Standards and
Control, Hertfordshire, UK
THERÉSE KALLUR • BioLamina, Stockholm, Sweden
LUCY KRAVETS • Centre for Blood Cell Therapies, Peter MacCallum Cancer Centre, East
Melbourne, Australia
ALBERTO LABARGA • Department of Computer Science and Artificial Intelligence,
University of Granada, Gardana, Spain
TENNEILLE E. LUDWIG • WiCell Research Institute, Madison, WI, USA
ANGEL G. MARTIN • StemTek Therapeutics, Derio, Spain
DIANNE NICOL • Centre for Law and Genetics, Faculty of Law, University of Tasmania,
Hobart, TAS, Australia
ANDRÉS PAVÓN • StemTek Therapeutics, Derio, Spain
TEIJA PEURA • Genea Biomedx, Sydney, NSW, Australia
MAHENDRA RAO • New York Stem Cell Foundation Research Institute, New York, NY,
USA; Q Therapeutics, Salt Lake City, UT, USA; Wake Forest Institute for Regenerative
Medicine, Wake Forest University, Winston-Salem, NC, USA

ix

x

Contributors

JOY RATHJEN • School of Medicine, University of Tasmania, Hobart, TAS, Australia
PETER RATHJEN • The Menzies Institute of Medical Research, University of Tasmania,
Hobart, TAS, Australia
KRISHANU SAHA • Department of Biomedical Engineering, University of
Wisconsin-Madison, Madison, WI, USA; Wisconsin Institute for Discovery,
University of Wisconsin-Madison, Madison, WI, USA
JUAN M. SALCEDO • StemTek Therapeutics, Derio, Spain
PAUL DE SOUSA • Roslin Cell Sciences Ltd., Midlothian, UK; Censo Biotechnologies Ltd.,
Midlothian, UK; Centre for Clinical Brain Sciences, University of Edinburgh,
Edinburgh, UK
GLYN STACEY • UK Stem Cell Bank, National Institute for Biological Standards and
Control, Hertfordshire, UK
SONYA STENFELT • Department of Neuroscience, Uppsala University, Uppsala, Sweden
EVA TOMASKOVIC-CROOK • AIIM Facility, ARC Centre of Excellence for Electromaterials
Science, Intelligent Polymer Research Institute, University of Wollongong, Fairy Meadow,
NSW, Australia; Illawarra Health and Medical Research Institute, University of
Wollongong, Wollongong, NSW, Australia
CESAR TRIGUEROS • StemTek Therapeutics, Derio, Spain
KRISTIAN TRYGGVASON • BioLamina, Stockholm, Sweden
LOREA ZABALETA • StemTek Therapeutics, Derio, Spain


Part I
Generic Themes in Stem Cell Banking

Chapter 1
Stem Cell Banking: A Global View
Glyn Stacey
Abstract
Stem cell banking has been a topic of discussion and debate for more than a decade since the first public
services to supply human embryonic stem cells (hESCs) were established in the USA and the UK. This
topic has received a recent revival with numerous ambitious programmes announced to deliver large collections of human induced pluripotency cell (hiPSC) lines. This chapter will provide a brief overview charting the development of stem cell banks, their value, and their likely role in the future.
Key words Pluripotent stem cell banks, Human embryonic stem cells, Induced pluripotent stem cells,
Rationale, History, Challenges

1

The Rationale for Stem Cell Banks
In all research using cell lines the scientific quality of the source
cells is crucial. The exchange of cell lines between researchers is
part of the traditional scientific currency securing inter-laboratory
collaboration. However, all too often cells exchanged in this way
have become genetically altered during culture passage, switched
or cross-contaminated with another cell line, or contaminated with
mycoplasma, which often leads to permanent adverse genetic and/
or phenotypic change [1]. The consequences for research performed with the wrong, altered, and/or mixed cell cultures are
clearly serious for the validity of any resulting published work and
can lead to retraction of publications. While the originators of cell
lines may pay special attention to supply suitable cells to collaborators, it has been shown in cancer cell lines that a significant proportion of cells volunteered for deposit in public collections are no
longer the original cell line (i.e., switched with another cell line)
and more seriously, numerous examples were provided by the originators of the lines themselves [2]. Already, examples of cross-contaminated and mycoplasma contaminated cells have been identified
among the hESCs available for research and in other cases the cell
line has become overgrown by a chromosomally abnormal clone.

Jeremy M. Crook and Tenneille E. Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol. 1590,

DOI 10.1007/978-1-4939-6921-0_1, © Springer Science+Business Media LLC 2017

3


4

Glyn Stacey

In recent work to establish the European bank of iPSC lines (www.
ebisc.org/), early submissions were exposed to comprise 14% hiPSCs lines that were not from the correct donor (J Holder, personal
communication). This situation requires all researchers to take
responsibility for assuring the characteristics of the cells they use,
and is a driver for the existence of resource centers focused on the
establishment of quality controlled seed stocks that will provide for
long-term supply of stem cell lines for research.
Centers dedicated to the long-term supply of stem cell lines
provide the opportunity for researchers to secure a number of additional benefits. These include safe backup stocks, access to advice,
and training in the culture and preservation of a range of different
cell lines and in some cases such as the UK Stem Cell Bank (SCB) a
patent depositary. A further and significant benefit from stem cell
banking is the assurance of good practice. A network of stem cell
banking centers and individuals and organizations committed to
supporting formalized stem cell banking called the International
Stem Cell Banking Initiative (ISCBI) has produced a consensus on
principles of best practice in the procurement, banking testing, and
distribution of hESC lines for research [3]. ISCBI has coordinated
international opinion on requirements for high-quality hiPSC
banking (2012 meeting report: />initiatives/international-stem-cell-banking-initiative/) and completed a consensus on development of seed stock of pluripotent
stem cell lines for clinical application [4].

Unfortunately, the technical challenge and cost of banking
cells has meant that operating such collections, of even the most
readily expanded cell lines, means that such resources do little
more than recover costs and require ongoing institutional subsidy
to sustain them (for example, banking a research-grade and clinicalgrade cell line costs approximately £60 thousand and £1 million
respectively when all staff, facilities, safety testing, and overheads
costs are accounted for). This is demonstrated by the very limited
number of banks engaged in the supply of human pluripotent stem
cell (hPSC) lines internationally at any significant level. Significant
improvements including advanced technologies for increased efficiencies in cell culture will be required to make self-supporting
stem cell banking a reality.

2

History of Banking Human Pluripotent Stem Cell Lines
The discovery that hESCs could be generated from human blastocysts in 1998 rightly caused great excitement. As pluripotent cells
can give rise to cells of the three germ layers required to form the
tissues of the human body, they gave hope to providing broad


Stem Cell Banking: A Global View

5

ranging restorative therapy to replace damaged or diseased tissue
[5]. This potential was immediately recognized by the US National
Institutes of Health (NIH) who funded the providers of lines to
supply them worldwide. Following UK legislation in 2001 to enable
the generation and sharing of hESC lines between researchers, the
Medical Research Council (MRC) coordinated a project to establish regulatory oversight and a national bank to ensure that stocks

of hESCs were made available from a single center. In due course,
the NIH sponsored centralized supply from the US pluripotent
stem cell bank at WiCell in Wisconsin ( />Although this funding has not been sustained, the operation has
been maintained by WiCell. In the UK, the UKSCB has secured
sustained government support via the MRC and Biotechnology
and Biological Sciences Research Council (BBSRC) and currently
operates a multi-sponsor operation with engagement in research
grants and increasing core support from its host organization the
National Institute for Biological Standards and Control (NIBSC),
now part of the UK regulatory body the Medicines and Healthcare
Products Regulatory Agency (MHRA). In the meantime, other
Government-funded and commercial suppliers of stem cell lines
have been established, and a number of institutions have supported
centers to focus on hiPSC generation and banking (e.g., Rutgers
hiPSC bank, EBiSC, Coriell). Of course, there are many core facilities providing local supplies of stem cells and a few companies who
can provide cells (e.g., Biotime, Cellartis (Takara)) but the number
of public service collections has remained relatively small primarily
due to the reasons outlined above. An additional challenge for
hESC collections has been the ethical debate over the use of human
embryos for research, which requires careful management to assure
the banks operate in an ethically responsible and neutral way.
Since the advent of hiPSC technology in 2009 [6], the ability
to generate stem cell lines using a relatively simple and readily
accessible technique from many kinds of tissue has resulted in a
great increase in the number of iPSC lines. Furthermore, the
capacity to generate lines from individuals with bespoke genotypes
and disease states has led to the development of major programmes
of work to isolate large numbers of lines from patients with inherited disease and other disease states (www.stembancc.org) even to
capture human biological diversity (www.hipsci.org). Details of the
major hiPSC operations are given in Table 1.

These initiatives are currently at an early stage but ultimately,
will deliver a significant resource for research and drug development. There are calls for these efforts to be coordinated to make
best use of the resources available and establish common standards,
in part based on the experience and expertise that has been developed by the stem cell banking field [7].


Rutgers University, USA
Grenada, Spain
Hsinchu, Taiwan
Hertfordshire, UK
WiCell Research Institute/Wisconsin, USA

RCUDR-Infinite Biologics

Spanish Stem Cell Bank

Taiwan Stem Cell Bank

UK Stem Cell Bank

WISC Bank

Harvard University/Massachusetts, USA
Stanford University, California, USA
University of Connecticut/Connecticut, USA

Harvard Stem Cell Institute iPS Core Facility

Stanford Institute for Stem Cell Biology and
Regenerative Medicine

UCONN Stem Cell Core

La Jolla, USA

/>
/>
Genea

Maryland, USA

GlobalStem

/>Stem_Cell_Research

/>
Goteburg, Sweden

Cellartis (Tokara)

/>
/>
/>
/>
Reproductive Genetics Institute (Stemride International) Illinois, USA

Alameda, USA

Biotime

Commercial Banks

Harvard University/Massachusetts, USA

Harvard HUES Facility

/>
Boston Children’s Hospital/Massachusetts, USA />stem-cell-program-labs/the-hesc-core-facility/

/>
/>
c.firdi.org.tw//index.do

/>bancoandaluzdecelulasmadre/

www.rucdr.org

Website

Children’s Hospital Boston hESC Core Facility

Core Facilities (examples only)

Location

Distributor

Table 1
Examples of stem cell banks and core facilities

Stem Cell Banking: A Global View

3

7

Core Requirements for the Establishment of a Pluripotent Stem Cell Bank
There are at least three fundamental issues to address before setting up such a facility:
1. The first step is to clearly identify the primary purpose of the
bank; is it supply of research grade cells, clinical grade cells,
other reagents, training, or a combination of these.
2. Another key decision is whether the bank will operate locally
as a so-called core facility or aim to deliver cells to a much
broader geographical range of clients.
These key decisions will clearly identify the investment required
in staff, facilities, and other resources (for more detailed reviews,
see refs. 7 and 8).
Having set the remit and resources for the Bank, a suitable
infrastructure needs to be established, which should include:
1. An appropriate and robust governance framework. This will
usually comprise the normal institutional management procedures (e.g., health and safety, security, financial accountability)
and the mechanisms for assuring appropriate ethical review
processes (e.g., Institutional Review Board, Local Research
Ethics Committee) are in place to ensure all cell lines are
proven to be isolated from tissues taken with fully informed
and appropriate consent for the distribution of derived cell
lines for any kind of research. In addition, it is important to
have high-quality scientific input by way of an external scientific advisory board or committee to ensure that a bank maintains standards and procedures fit for current research need.
2. A system of quality assurance that is suitable for the intended

purpose of the bank and focused on the needs of users groups.
Key to developing this will be the standard of operation set at
the outset (above) as supply for research as opposed to clinical
use will require very different levels of assurance and scrutiny
and compliance with specific quality standards. This will
typically involve the preparation of a high-level operational
manual including key policies and standards and also
documenting all key procedures and protocols and appropriate
record keeping to assure the required level of traceability. This
QA system does not need to be under a formal quality standard
as supply to local researchers could be operated with a minimal
system that suits the clients involved.
The quality assurance system should also include a Cell Line
Master File (employed by the UKSCB) or Cell Line History File as
ref. [3] a key element. This is valuable for both research grade an
clinical grade seed stocks [9, 10], as it can be used to capture all
information from details of informed consent through to quality


8

Glyn Stacey

control data and release of the cell line. Such information may be
difficult to gather retrospectively and will be a critical source of
information to assure appropriate levels of risk assessment, risk
mitigation, and regulatory acceptability of the cell line for clinical
application.

4

Technical Challenges for Stem Cell Banks
In order to deliver the large numbers of existing and future cell
lines, there is a critical need to enhance the efficiency of stem cell
banking and reduce costs. This will require significant developments in banking procedures and technology. Mechanization and
automation will be the key to this effort and a number of systems
are under development. Improvements to provide stem cell cultures that are consistent and have minimal levels of differentiated
cells will come from improvements in culture media and surface
treatments or 3D culture. In addition, new quality control and
characterization techniques will need to be developed to streamline release of cell banks and reduce costs. However, it will be
important to ensure that standards for acceptability of cell lines
are not compromised. Rapid array-based and next-generation
sequencing systems for screening for adventitious agents and
expression of key stem cell markers are already developing that
will need to be qualified for routine use. The use of teratoma
assays is incompatible with such an approach and there are in any
case, serious challenges for the reliability of this type of assay [11,
12] and a comparison of the variation in results fro these assays is
presented in the accompanying tables to ref. [3]. Already array
systems for determining epigenetic status have been proposed for
determining potential pluripotency [13]. However, such assays
will have to be carefully evaluated to ensure that such profiles are
closely correlated with pluripotent capability and will not include
non-pluripotent cell types. It is likely that a rapid cell culture assay
to measure directed lineage commitment will be needed in combination with epigenetic screens. An international collaboration
(the International Stem Cell Initiative; www.stem-cell-forum-net)
is currently comparing such methods with optimized directed differentiation protocols and the teratoma assay and is now preparing its final report (submitted for publication). Hopefully, studies
like this will enable a replacement regime for the teratoma assay to
be established that can enhance the quality and routine use of
pluripotency studies.

4.1 Current
and Developing Issues
for Stem Cell Banks

Ethical issues remain a challenge in this area. The concerns over
the use of human embryos to generate hESC lines are often said
to have been removed by using hiPSC lines. However, it is still
possible that gametes and thus embryos could be generated
in vitro for reproductive cloning using iPSC lines. Public


Stem Cell Banking: A Global View

9

concern is to be expected wherever work on gametes is proposed
out. The ability to guarantee donor anonymity is also an area for
careful consideration. Large data sets are now available with the
revolution in genomic analytical techniques such as deep sequencing. Two issues in particular arise from this. One is that researchers may be presented with an ethical dilemma should they make an
adverse discovery in a cell line. Appropriate procedures for deciding how to deal with such situations need to be considered carefully, with initial regard being given by the Ethics Working Party
of the International Stem Cell Forum [14]. Second, it has been
shown that it is possible to link data in databases with deidentified
genetic data with genealogy websites holding partial Y chromosome STR data for known individuals [15]. Many high-level ethical reviews have concluded that it is appropriate to make scientific
data broadly available to the research community. However, the
mechanisms to assure donors remain deidentified are not secure
against deliberate attempts to reidentify donors and solutions may
have to rely on researchers vigilance and honesty when signing up
to gain access to donor genetic data [16]. In the longer term stem
cell banks and others managing genetic data will need to understand that donor anonymity cannot be guaranteed and consents

must reflect this reality.
A further ethical issues that will challenge stem cell banks, the
whole stem cell field, and society in general are development of
animal-human chimeric tissues in animal models, the capacity of
iPSCs to generate gametes, and potentially embryos and cloned
individuals. More specifically for stem cell banks a further challenge will be the need to support application of stem cell lines in an
increasing spectrum of research applications.
Automation of stem cell culture processes will be vital to enable
the delivery of the current ambitious large-scale cell banking projects for iPSC lines. It is currently possible to automate elements of
the cell expansion and characterization tasks but a fully automated
system that does not require manual intervention between recovery
of a vial, its expansion and differentiation ready for final processing
for therapy is not yet qualified for routine clinical use. However,
some groups are moving toward such systems that may be available
for routine use in the future, e.g., Kawasaki, Hamilton, New York
Stem Cell Foundation (NYSCF), Tokyo Electron and Tecan.
Stem Cell research and cell therapy are dynamic and rapidly
progressing areas and it would be easy for stem cell banking centers
to be driven to respond to latest developments that may not be
fruitful in the long run and wasteful of bank resources. It is therefore crucial for each banking center to engage high-quality scientific advice that can be used to guide such centers to carry out
appropriate feasibility studies that enable the bank to make secure
decisions that will help to ensure that any major investment in new
technology is effective.


10

Glyn Stacey

References

1. Rottem S, Naot Y (1998) Subversion and
exploitation of host cells by mycoplasma.
Trends Microbiol 6:436–440
2. MacLeod RAF, Dirks WG, Matsuo Y et al (1998)
Widespread intraspecies cross-contamination of
human tumour cell lines arising at source. Int
J Cancer 83:555–563
3. Andrews PW, Baker D, Benvinisty N et al
(2015) Points to consider in the development
of seed stocks of pluripotent stem cells for clinical applications: International Stem Cell
Banking Initiative (ISCBI). Regen Med 10(2
Suppl):1–44
4. Andrews PW, Arias-Diaz J, Auerbach J et al
(2009) Consensus guidance for banking and
supply of human embryonic stem cell lines for
research purposes. Stem Cell Rev 5(4):301–314
5. Thomson JA, Itskovitz-Eldor J, Shapiro SS
et al (1998) Embryonic stem cell lines derived
from
human
blastocysts.
Science
282(5391):1145–1147
6. Nakagawa M, Koyanagi M, Tanabe K et al
(2008) Generation of induced pluripotent
stem cells without Myc from mouse and human
fibroblasts. Nat Biotechnol 26(1):101–106
7. Stacey G, Crook JM, Hei D, Ludwig T (2013)
Banking human induced pluripotent stem
cells: lessons learned from embryonic stem

cells? Cell Stem Cell 13(4):385–388
8. Inamdar MS, Healy L, Sinha A, Stacey G
(2012) Global solutions to the challenges of

9.

10.

11.

12.

13.

14.

15.

16.

setting up and managing a stem cell laboratory. Stem Cell Rev 8(3):830–843
Stacey G (2012) Banking stem cells for
research and clinical applications. Prog Brain
Res 200:41–58
Stacey G, Masters JR (2008) Cryopreservation
and banking of mammalian cell lines. Nat
Protoc 3(12):1981–1989
Buta C, David R, Dressel R et al (2013)
Reconsidering pluripotency tests: do we still
need teratoma assays? Stem Cell Res 11:

552–562
Müller FJ, Goldmann J, Löser P, Loring JF
(2010) A call to standardize teratoma assays
used to define human pluripotent cell lines.
Cell Stem Cell 6(5):412–414
Müller FJ, Schuldt BM, Williams R et al
(2011) A bioinformatic assay for pluripotency
in human cells. Nat Methods 8(4):315–317
Isasi R, Knoppers BM, Andrews PW et al
(2012) Disclosure and management of
research findings in stem cell research and
banking: policy statement. Regen Med 7(3):
440–448
Gymrek M, McGuire AL, Golan D et al (2013)
Identifying personal genomes by surname
inference. Science 339(6117):321–324
Isasi R (2014) Stem cell research and banking:
towards policy on disclosing research results
and incidental findings. In: Dusko I (ed) Stem
cell banking. Springer, New York, pp 29–40


Chapter 2
Quality Assurance in Stem Cell Banking: Emphasis
on Embryonic and Induced Pluripotent Stem Cell Banking
Therése Kallur, Pontus Blomberg, Sonya Stenfelt,
Kristian Tryggvason, and Outi Hovatta
Abstract
For quality assurance (QA) in stem cell banking, a planned system is needed to ensure that the banked
products, stem cells, meet the standards required for research, clinical use, and commercial biotechnological applications. QA is process oriented, avoids, or minimizes unacceptable product defects, and particularly encompasses the management and operational systems of the bank, as well as the ethical and legal

frameworks. Quality control (QC) is product oriented and therefore ensures the stem cells of a bank are
what they are expected to be. Testing is for controlling, not assuring, product quality, and is therefore a
part of QC, not QA. Like QA, QC is essential for banking cells for quality research and translational application (Schwartz et al., Lancet 379:713–720, 2012). Human embryonic stem cells (hESCs), as cells
derived from donated supernumerary embryos from in vitro fertilization (IVF) therapy, are different from
other stem cell types in resulting from an embryo that has had two donors. This imposes important ethical
and legal constraints on the utility of the cells, which, together with quite specific culture conditions,
require special attention in the QA system. Importantly, although the origin and derivation of induced
pluripotent stem cells (iPSCs) differ from that of hESCs, many of the principles of QA for hESC banking
are applicable to iPSC banking (Stacey et al., Cell Stem Cell 13:385–388, 2013). Furthermore, despite
differences between the legal and regulatory frameworks for hESC and iPSC banking between different
countries, the requirements for QA are being harmonized (Stacey et al., Cell Stem Cell 13:385–388,
2013; International Stem Cell Banking Initiative, Stem Cell Rev 5:301–314, 2009).
Key words Quality assurance, Quality control, Stem cell banks, Human embryonic stem cells,
Induced pluripotent stem cells

1

Introduction
The QA in stem cell banking ensures that the quality of the banked
stem cells is in accordance with national and international guidelines and standards for supply of stem cells for research, biotechnological applications, and/or clinical use [1–3]. As such, a bank must
identify the purpose of banked cells to comply with the correct QA
standard to cover the range of potential applications. There must be
a continuous process of managing and evaluating the QA system for

Jeremy M. Crook and Tenneille E. Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol. 1590,
DOI 10.1007/978-1-4939-6921-0_2, © Springer Science+Business Media LLC 2017

11

12

Therése Kallur et al.

Fig. 1 Schematic of quality management in a stem cell bank

total quality management including risk assessments of all aspects of
stem cell banking (Fig. 1). It includes processes of selection of the
cells donors, the ethical processes around the specific cells banked,
training of the personnel of the bank, the quality and function of
the equipment in the bank, the materials used in culture and freezing of the cells, documentation in the bank, process and material
release, the regulatory standards that are implemented (e.g.,
International Standards Organization; ISO) [4], and the accepted
guidelines and principles in stem cell banking, such as the
International Society for Stem Cell Research (ISSCR) Guidelines
for the conduct of hESC research [5], International Stem Cell
Banking Initiative (ISCBI) Guidance for Banking [1], OECD
Good Laboratory Practice principles [6], as well as appropriate QC.

2

Quality Systems and Standards
Good Laboratory Practice (GLP) regulatory framework is applied
for research grade hESCs, iPSCs, and tissue-derived stem cells
intended for safety studies in animals, while good Manufacturing
Practice (GMP) is required for all clinical grade cells. GLP and


Quality Assurance in Stem Cell Banking…

13

GMP include the quality of management and distribution of the
cell lines as well as the quality of the cells. Of fundamental importance is that the general ethics principles regarding consent and
donation of human cells and tissue as well as for stem cell banking
have been followed and documented. The cell processing technology, management, and running system used in the bank have to be
quality assured. Procedures and protocols for cell culture, freezing,
and thawing must be described and results from cell characterization including the ability of the cells to differentiate, their functionality, microbial testing, and tumorigenicity studies must be
sufficiently documented. The bank must have a system for managing the inventory for assuring credible and accurate storage of
every cell vial, showing the location and number of vials, and who
was responsible for freezing, removing, etc. In addition, specified
testing procedures should be performed before the release of any
cell vials to research and/or clinical recipients.
Examples of QA for clinical grade human embryonic stem cells
have been described previously in the literature [7, 8]. General
standards for QA are applicable to stem cell banking, including
those from the ISO. Such standards are:
1. ISO9001:2000, a general quality management standard for
provision of services and products;
2. ISO17025, laboratory testing and monitoring including the
cell lines for testing of medical products;
3. ISO13485, diagnostic testing procedures including the use of
cells or cell-derived reference materials;
4. ISO34, guide for preparation of reference materials.
The processes and features of stem cell banking requiring QA
are listed in Table 1 and Fig. 2.
Table 1
QA in stem cell banking
Management
Principles of ethical and legal requirements, consents, and agreements

Procedures for receiving, expanding, and storing of stem cells
Operation and maintenance of equipment
Characterization of cells:
(A) Quality and purity of the cell population
(B) Functionality of the cells
• Pluripotency of hESC and hiPSC
• Microbial testing
• Genetic testing
• Tumorigenicity
Release criteria of the cells
Transport and distribution of the cells


14

Therése Kallur et al.

Fig. 2 A flow chart of QA in a hESC bank

3

Consents and Legal Framework
Despite several sets of guidelines, the main principles for QA of
stem cell banks are similar in most European countries, North
America, and Australia. The origin of cells is important, together
with the agreements for storage and conditions for distribution.
The stem cell type and the purpose for using the cells varies but the
stem cell bank has to ensure consent by the donating person/persons is voluntary, has been well documented, and guarantees the
privacy of the donor information. Release and distribution of stem
cells should only be possible for projects that are approved by ethics committee and never to any third parties without appropriate

permissions. Commercial and clinical use of the donated stem cells
requires particular ethics and legal agreements that follow the legislation of the country of origin and the country of banking. There
are particular laws for the use of hESCs in almost all countries. The
European laws are presented by European Science Foundation [9,
10]. In addition, the bank should comply with guidelines established by the ISSCR [5] which include ethics principles and also
national guidelines such as in the USA [11, 12] (US National
Academy of Science, NAS 2005, NIH Guidelines for Human
Embryonic Stem Cell Research ) and
other relevant regulatory authorities.


Quality Assurance in Stem Cell Banking…

4

15

QA Is Necessary for Stem Cell Banking Activities
In order to ensure the banking and provision of good quality cells,
cell lines must be qualified by mandatory QC procedures for the
banks QA. For example, effective and safe stem cell scaleup/expansion methods must be employed to avoid culture adaptation or tumorigenic mutations. Avoiding animal-derived components
in the cultures or feeder cells is a preferred strategy to humanize and
simplify processing. If feeder cells are used, they should be of human
origin and ideally GMP or GLP grade depending on the sownstream application [13, 14]. It is advisable to keep the passage level
of the banked stem cells as low as possible in the master cell bank
(MCB), which maximizes up-scaling and supply of more cells of a
particular passage. Furthermore, it is advisable that the stem cell
bank use cell cultivation methods that are considered current best
practice. According to our experience, the use of human recombinant laminin-521 as culture substrates and chemically defined culture media allow the effective expansion of hESCs [15].
Importantly, optimized phenotype characterization methods

for each stem cell type are essential, including testing for pluripotency in vivo by teratoma test in immune-compromised mice, and
in vitro by differentiating them into mesoderm, endoderm, and
ectoderm derivatives. In order to confirm pluripotency, cells should
exhibit typical cell surface marker profile by the expression of relevant proteins suing, for example, flow cytometry and
immunocytochemistry.
Exclusion of tumorigenic changes and abnormal karyotype is
an essential part of safety testing. This can be done using standard
G-band analysis, comparative genomic hybridization (CGH), and
single nucleotide polymorphism (SNP) testing [16–19] but also by
high-throughput sequencing.
Microbial testing is also an important part of QC and the
release criteria encompassed by the bank QA. Microbial contamination not only affects the quality of the research data obtained
from a particular cell line but also poses a risk for staff at the bank.
The QA system must include test criteria for cell donors, cell lines,
and reagents and biological material used for cell culture (see
Chapter 6).
An important part of running a cell bank is ensuring that the
quality and identity of the cells are maintained during the transport
and distribution of the cells to and from the bank. This should
include having standard operating procedures (SOPs) in place for
temperature control, as wells as for record keeping including deposit
and withdrawal or requisition of cells. For cells intended for clinical
use, a guidance for how such a system may be established can be
found in the EU guidelines on Good Distribution Practice (GDP)
of medicinal products for human use [20]. Stem cells can be used
in research or in clinical treatment. Given the many different stem


16

Therése Kallur et al.

cell types and grades, it is important for the end users to know
exactly which stem cells are being provided for their particular purpose. There are specific requirements and standards for cells for
both research and clinical use, including sterility, functionality,
genetic stability, lack of immunogenic substances, traceability of the
culture constituents, in addition to traceability of the cells and cell
donors. Ensuring the requirements are met will ensure safety, quality, efficacy, and reproducibility.
References
1. International Stem Cell Banking Initiative
(2009) Consensus guidance for banking and
supply of human embryonic stem cell lines for
research purposes. Stem Cell Rev 5:301–314
2. Crook JM, Hei D, Stacey G (2010) The
International Stem Cell Banking Initiative
(ISCBI): raising standards to bank on. In Vitro
Cell Dev Biol Anim 46:169–172
3. Healy L, Young L, Stacey GN (2011) Stem
cell banks: preserving cell lines, maintaining
genetic integrity, and advancing research.
Methods Mol Biol 767:15–27
4. The
International
Organization
for
Standardization. www.iso.org
5. Guidelines for the Conduct of Human
Embryonic Stem Cell Research (2005) www.
ISSCR.org
6. OECD (2013) Principles of good laboratory

practice and compliance monitoring. www.
OECD.org
7. Crook JM, Peura TT, Kravets L et al (2007)
The generation of six clinical grade human
embryonic stem cell lines. Cell Stem Cell
1:490–494
8. Ilic D, Stephenson E, Wood V (2012)
Derivation and feeder-free propagation of
human embryonic stem cells under xeno-free
conditions. Cytotherapy 14:122–128
9. Hovatta O., Walles H., Agovic A. et al.
(2010) Human stem cell research and
regenerative medicine: European perspective on scientific, ethical and legal issues.
ESF Science Policy Briefing 38. http://
www.esf.org/publications/science-policybriefings.html
10. Hovatta O, Stojkovic M, Nogueir M, VarelaNieto I (2010) European scientific, ethical and
legal issues on human stem cell research and
regenerative medicine. Stem Cells 28:1005–1007
11. US National Academy of Science (2005)
Guidelines for Human Embryonic Stem Cell
Research. www.nap.edu

12. National Institutes of Health (NIH) Guidelines
for Human Embryonic Stem Cell Research.

13. Unger C, Skottman H, Blomberg P et al
(2008) Good manufacturing practice and clinical grade human embryonic stem cell lines.
Hum Mol Genet 17(R1):R48–R53
14. Prathalingam N, Ferguson L, Young L (2012)
Production and validation of a good manufacturing practice grade human fibroblast line for

supporting human embryonic stem cell derivation and culture. Stem Cell Res Ther
3(2):12
15. Rodin S, Antonsson L, Niaudet C et al (2014)
Clonal culturing of human embryonic stem
cells on laminin-521/E-cadherin matrix in
defined and xeno-free environment. Nat
Commun 5:3195
16. Hovatta O, Jaconi M, Töhönen V et al (2010)
A teratocarcinoma-like human embryonic
stem cell (hESC) line and four karyotypically
normal hESC lines reveal high oncogenic
potential. PLoS One 23(5):e10263
17. Närvä E, Autio R, Rahkonen N et al (2010)
High resolution genome wide DNA analysis
on a large panel of human embryonic stem
cells reveals novel genomic changes associated
with alterations in gene expression. Nat
Biotechnol 28(4):371–377
18. International Stem Cell Initiative (2011)
Screening ethnically diverse human embryonic
stem cells identifies a chromosome 20 minimal
amplicon conferring growth advantage. Nat
Biotechnol 29:1132–1144
19. Stephenson E, Ogilvie CM, Patel H et al
(2010) Safety paradigm: genetic evaluation of
therapeutic grade human embryonic stem
cells. J R Soc Interface 7(Suppl 6):S677–S688
20. Information from European Union institutions, bodies, offices and agencies. Other Acts.
Guidelines of 7 March 2013 on good distribution practice of medicinal products for human
use (2013/C 68/01)

Chapter 3
Acquisition and Reception of Primary Tissues, Cells,
or Other Biological Specimens
Lyn E. Healy
Abstract
The use and banking of biological material for research or clinical application is a well-established practice.
The material can be of human or non-human origin. The processes involved in this type of activity, from
the sourcing to receipt of materials, require adherence to a set of best practice principles that assure the
ethical and legal procurement, traceability, and quality of materials.
Key words Tissue, Cells, Biospecimens, Procurement, Consent, Quality, Best practice

1

Introduction
There is a wide variety of biological material used in the processing
and banking of stem cells. However, not all material is of human
origin and not all contains viable material. This chapter primarily
focuses on human material but also considers other sources of
material frequently used both in and ancillary to the banking process. The sourcing, procurement, acquisition, and receipt of human
biological material for research or clinical application form the initial key stages in the activities of stem cell banking and bioprocessing [1–3]. The material may be used for a number of activities:
hemopoietic stem cells (HSC) for clinical application [4], various
sources of tissue as starting material for the generation of cell lines,
including induced pluripotent stem cells (iPSCs) [5, 6], other biological material for example extracted nucleic acids used for controls
in routine molecular biology. Ensuring that cells, tissues, cell lines,
and other biological material are sourced and procured ethically and
in accordance with the local legal framework underpins the operating principles of any reputable biorepository and the activity of stem
cell banks falls within this category of repository. Human stem cell
banks are repositories that either specialize in the banking of a particular cell type or bank a number of cell types including somatic

primary cells (e.g., cord blood, mesenchymal, embryonic stem cells,

Jeremy M. Crook and Tenneille E. Ludwig (eds.), Stem Cell Banking: Concepts and Protocols, Methods in Molecular Biology, vol. 1590,
DOI 10.1007/978-1-4939-6921-0_3, © Springer Science+Business Media LLC 2017

17