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 M E T H O D S I N M O L E C U L A R B I O L O G Y™

Cryopreservation
and Freeze-Drying
Protocols
Second Edition

Edited by

John G. Day
Culture Collection of Algae and Protozoa
Scottish Association for Marine Science
Dunstaffnage Marine Laboratory
Dunbeg, Argyll, UK

Glyn N. Stacey
Division of Cell Biology and Imaging
National Institute for Biological Standards and Control
Potters Bar, Herts., UK

Day_FM_Final 3 4/24/07, 2:31 PM

 © 2007 Humana Press Inc.
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Library of Congress Cataloging-in-Publication Data
Cryopreservation and freeze-drying protocols / edited by John G.
Day, Glyn N. Stacey. -- 2nd ed.
p. ; cm. -- (Methods in molecular biology ; 368)
Includes bibliographical references and index.
ISBN 1-58829-377-7 (alk. paper)
1. Cryopreservation of organs, tissues, etc.--Methods. 2.
Freeze-drying--Methods. I. Day, John G., 1961- II. Stacey, G.
(Glyn) III. Series: Methods in molecular biology (Clifton, N.J.) ;
v. 368.
[DNLM: 1. Cryopreservation--methods. 2. Freeze Drying--methods.
W1 ME9616J v.368 2007 / QY 95 C957 2007]
QH324.9.C7C77 2007
570.75'2--dc22 2006015414

Day_FM_Final 4 4/24/07, 2:31 PM

 Preface
The preservation of biological material in a stable state is a fundamental
requirement in biological/medical science, agriculture, and biotechnology.
It has enabled standardization of experimental work over time, has secured
lifesaving banks of cells and tissue ready for transplantation and transfusion at
the time of need and has assured the survival of critical germ plasm in support
of programs for the conservation of species. Cryopreservation and freeze-
drying are widely accepted as the preferred techniques for achieving long-term
storage, and have been applied to an increasingly diverse range of biological
materials. Although the basis for many methodologies is common, many laborato-
ries lack expertise in applying correct preservation and storage procedures and
many apply outdated or inappropriate protocols for storing samples or cultures.
Cryopreservation and Freeze-Drying Protocols, Second Edition, in addi-
tion to outlining the fundamental principles associated with the conservation
of biological resources, freeze-drying, and cryopreservation, is a compilation
of cryopreservation and freeze-drying methodologies applicable to different
biological materials, which have been developed by expert laboratories. The
protocols are reproducible, robust, and most have been transferred success-
fully to other laboratories. Our intended readers are those proposing to estab-
lish, or improve, biostorage systems in their laboratory, whether concerned
with biological resource centers, animal husbandry, aquaculture, horticulture,
medicine, or human fertilization programs.
Because the emphasis of this volume is on methodology it is intended that
practical progress can be made without reference to other sources. Each chap-
ter deals with a discrete biological resource: a short introduction on the status
of biostorage development is followed by a detailed description of materials
required and methodological protocol to be followed, with explanatory notes
on key technical issues.
This second edition expands on the range of materials covered in the 1995
edition and includes many novel approaches and protocols for biological mate-
rials that were not preservable in 1995. However, there are still many materials
that remain preservation-recalcitrant, we hope and trust that future editions
will contain cryopreservation and freeze-drying protocols that can be used to
preserve biological resources that are at present recalcitrant to successful pres-
ervation.
John G. Day
Glyn N. Stacey

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Day_FM_Final 6 4/24/07, 2:31 PM
 Contents
Preface .............................................................................................................. v
Contributors .....................................................................................................ix
Glossary of Specialized Terms .........................................................................xi

1 Long-Term Ex Situ Conservation of Biological Resources

and the Role of Biological Resource Centers .................................... 1
Glyn N. Stacey and John G. Day
2 The Principles of Freeze-Drying .......................................................... 15
Gerald Adams
3 Principles of Cryopreservation ............................................................ 39
David E. Pegg
4 Lyophilization of Proteins ................................................................... 59
Paul Matejtschuk
5 Vacuum-Drying and Cryopreservation of Prokaryotes ........................ 73
Brian J. Tindall
6 Freeze-Drying of Yeast Cultures .......................................................... 99
Chris Bond
7 Cryopreservation of Yeast Cultures ................................................... 109
Chris Bond
8 Freeze-Drying Fungi Using a Shelf-Freeze Drier ............................... 119
C. Shu-hui Tan, Cor W. van Ingen, and Joost A. Stalpers
9 Cryopreservation and Freeze-Drying of Fungi Employing
Centrifugal and Shelf Freeze-Drying ............................................. 127
Matthew J. Ryan and David Smith
10 Cryopreservation of Microalgae and Cyanobacteria ......................... 141
John G. Day
11 Cryopreservation of Plant Cell Suspensions ...................................... 153
Brian W. W. Grout
12 Cryopreservation of Shoot Tips and Meristems ................................. 163
Erica E. Benson, Keith Harding, and Jason W. Johnston
13 Cryopreservation of Desiccation-Tolerant Seeds............................... 185
Hugh W. Pritchard
14 Cryopreservation of Fish Sperm ........................................................ 203
Eugeny Kopeika, Julia Kopeika, and Tiantian Zhang

vii

Day_FM_Final 7 4/24/07, 2:32 PM

 viii Contents

15 Cryopreservation of Avian Spermatozoa ........................................... 219

Graham J. Wishart
16 Cryopreservation of Animal and Human Cell Lines .......................... 227
Christopher B. Morris
17 Cryopreservation of Hematopoietic Stem/Progenitor Cells
for Therapeutic Use ....................................................................... 237
Suzanne M. Watt, Eric Austin, and Sue Armitage
18 Cryopreservation of Human Embryonic Stem Cell Lines .................. 261
Charles J. Hunt and Paula M. Timmons
19 Cryopreservation of Primary Animal Cell Cultures ........................... 271
Glyn N. Stacey and Stuart Dowall
20 Cryopreservation of Red Blood Cells and Platelets ........................... 283
Andreas Sputtek
21 Cryopreservation of Mammalian Semen ........................................... 303
Mark R. Curry
22 Cryopreservation of Mammalian Oocytes ......................................... 313
Sharon J. Paynter and Barry J. Fuller
23 Cryopreservation of Mammalian Embryos ........................................ 325
Barry J. Fuller and Sharon J. Paynter
Index ............................................................................................................ 341

Day_FM_Final 8 4/24/07, 2:32 PM

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 Contributors
SUE ARMITAGE • National Blood Service-Oxford and Manchester, NHS Blood
and Transplant Authority, Edgware, UK
ERIC AUSTIN • National Blood Service-Oxford and Manchester, NHS Blood
and Transplant Authority, Manchester, UK
GERALD ADAMS • Lyosolutions, Salisbury, Wiltshire, UK
ERICA E. BENSON • DAMAR, Research Scientists, Conservation, Environmental
Science and Biotechnology, Cupar, Fife, Scotland, UK
CHRIS BOND • National Collection of Yeast Cultures, BBSRC Institute
of Food Research, Colney, Norwich, UK
MARK R. CURRY • Department of Biological Sciences, University of Lincoln,
Lincoln, UK
JOHN G. DAY • Culture Collection of Algae and Protozoa, Scottish
Association for Marine Science, Dunstaffnage Marine Laboratory,
Dunbeg, Argyll, UK
STUART DOWALL • European Collection of Animal Cell Cultures, Health
Protection Agency, Centre for Emergency Preparedness
and Response, Salisbury, Wiltshire, UK
BARRY J. FULLER • University Department of Surgery, Royal Free
and University College Medical School, London, UK
BRIAN W. W. GROUT • Post Graduate School, Writtle College, Chelmsford,
Essex, UK
KEITH HARDING • DAMAR, Research Scientists, Conservation, Environmental
Science and Biotechnology, Cupar, Fife, Scotland UK
CHARLES J. HUNT • UK Stem Cell Bank, Division of Cell Biology
and Imaging, National Institute for Biological Standards and Control,
South Mimms, Hertfordshire, UK
JASON W. JOHNSTON • HortResearch, Mt Albert, Auckland, New Zealand
EUGENY KOPEIKA • Institute for Problems of Cryobiology and Cryomedicine
of the National Academy of Science of the Ukraine, Kharkov, Ukraine
JULIA KOPEIKA • Luton Institute of Research in the Applied Natural Sciences,
University of Luton, Luton, Bedfordshire, UK
PAUL MATEJTSCHUK • Standardization Science, National Institute of Biological
Standards and Control, Potters Bar, Hertfordshire, UK
CHRISTOPHER B. MORRIS • European Collection of Animal Cell Cultures,
Health Protection Agency, Centre for Emergency Preparedness
and Response, Salisbury, Wiltshire, UK
ix

Day_FM_Final 9 4/24/07, 2:32 PM

 x Contributors

SHARON J. PAYNTER • Department of Obstetrics and Gynaecology, Wales

College of Medicine, Cardiff University, Cardiff, UK
DAVID E. PEGG • Medical Cryobiology Unit, Department of Biology,
University of York, York, UK
HUGH W. PRITCHARD • Jodrell Laboratory, Royal Botanic Gardens Kew,
Ardingly, West Sussex, UK
MATTHEW J. RYAN • Genetic Resources Collection, CABI Bioscience,
Egham, Surrey, UK
DAVID SMITH • Genetic Resources Collection, CABI Bioscience, Egham,
Surrey, UK
GLYN N. STACEY • Division of Cell Biology and Imaging, National Institute
for Biological Standards and Control, Potters Bar, Hertfordshire, UK
JOOST A. STALPERS • Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands
ANDREAS SPUTTEK • Universitätsklinikum Hamburg-Eppendorf, Institut für
Transfusionsmedizin, Hamburg, Germany
C. SHUI-HUI TAN • Centraalbureau voor Schimmelcultures, Utrecht,
The Netherlands
PAULA M. TIMMONS • UK Stem Cell Bank, Division of Cell Biology
and Imaging, National Institute for Biological Standards and Control,
South Mimms, Hertfordshire, UK
BRIAN J. TINDALL • DSMZ-Deutsche Sammlung von Mikroorganismen
und Zellkulturen GmbH, Braunschweig, Germany
COR W. VAN INGEN • Netherlands Vaccin Institute, AL Bilthoven,
The Netherlands
SUZANNE M. WATT • National Blood Service-Oxford and Manchester, NHS
Blood and Transplant Authority, Oxford, UK
GRAHAM J. WISHART • School of Contemporary Sciences, University
of Abertay Dundee, Dundee, UK
TIANTIAN ZHANG • Luton Institute of Research in the Applied Natural
Sciences, University of Luton, Luton, Bedfordshire, UK

Day_FM_Final 10 4/24/07, 2:32 PM

 Glossary of Specialized Terms
Cell Packing Effect: The effect on the survival of cells when frozen at a high, rather
than a low, packing density.
Chilling Injury: Injury that occurs as a result of a reduction in temperature.
Colligative Effect: A physical property of a system that depends on the number of
molecules and not their nature.
Cryopreservation: The storage of a living organism, or a portion thereof, at an ultra-
low temperature (typically colder than –130°C) such that it remains capable of
survival upon thawing.
Cryoinjury: Damage caused by reduction in temperature irrespective of the mechanism.
Cryoprotectant or Cryoprotective Agent (CPA): A substance that protects a living
system against injury due to reduction in temperature.
Cryostorage: The storage of a living organism, or a portion thereof, at an ultra-low
temperature (typically colder than –130°C) such that it remains capable of
survival upon thawing.
Eutectic Temperature: The lowest temperature (for a crystallizing solute) at which
the existence of a liquid phase for a given system is possible.
Freezing: The crystallization of liquid water to form ice.
Freeze-Drying: A controllable method of dehydrating labile products by vacuum des-
iccation (also termed as lyophilization).
Glass Transition Temperature (Tg'): The temperature (for an amorphous solute)
where the residual liquid vitrifies in the presence of ice.
Intracellular Freezing: The formation of ice crystals within cells.
Lyophilization: A controllable method of dehydrating labile products by vacuum
desiccation (also termed “freeze-drying”).
Melting Point: The temperature during the warming of an aqueous system at which
the last ice melts. This temperature is equal to the equilibrium freezing point.
Nucleation: The formation of a nucleus upon which an ice crystal can grow; this may
be an appropriate arrangement of water molecules or a foreign particle.
Solution Effects: Damage to cells that is a result of the increase in solute concentra-
tion that occurs as a secondary effect of freezing.
Super-Cooling: Reduction of temperature below the equilibrium freezing point but
without freezing, hence, an unstable situation. Note: supercooling is often not
hyphenated.
Vitrification: The conversion of an aqueous system to an amorphous, noncrystalline
solid solely by increase in viscosity.

xi

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Day_FM_Final 12 4/24/07, 2:32 PM
01_Day 4/17/07 10:32 AM Page 1

1
Long-Term Ex Situ Conservation of Biological Resources
and the Role of Biological Resource Centers
Glyn N. Stacey and John G. Day

Summary
The establishment and maintenance of biological resource centers (BRCs) requires careful
attention to implementation of reliable preservation technologies and appropriate quality
control to ensure that recovered cultures and other biological materials perform in the same
way as the originally isolated culture or material. There are many types of BRC that vary both
in the kinds of material they hold and in the purposes for which the materials are provided. All
BRCs are expected to provide materials and information of an appropriate quality for their
application and work to standards relevant to those applications. There are important industrial,
biomedical, and conservation issues that can only be addressed through effective and efficient
operation of BRCs in the long term. This requires a high degree of expertise in the maintenance
and management of collections of biological materials at ultra-low temperatures, or as freeze-
dried material, to secure their long-term integrity and relevance for future research, develop-
ment, and conservation.

Key Words: Biological resource centers; preservation; microorganisms; cell lines; tissues.

1. Introduction
Collecting examples of different types of organisms has been the pursuit of
scientists and amateur collectors for centuries. This activity was originally stim-
ulated by man’s curiosity regarding the natural diversity of “his” environment,
but for well over a century scientists have been collecting strains of animals,
plants, and microorganisms with specific scientific and technical aims relating
to taxonomy, infectious disease, and biochemistry. The first collection of
microorganisms for industrial use was established by Kral in 1869 and collec-
tions of plants and other organisms developed based on the maintenance of
examples of each strain or species under controlled laboratory or field conditions.

From: Methods in Molecular Biology, vol. 368: Cryopreservation and Freeze-Drying Protocols, Second Edition
Edited by: J. G. Day and G. N. Stacey © Humana Press Inc., Totowa, NJ

1
01_Day 4/17/07 10:32 AM Page 2

2 Stacey and Day

However, such collections of actively growing cultures suffered from complica-

tions, such as adaptation of the organism to the in vitro environment, genetic
mutations, contamination, and accidental loss of cultures. Clearly a mechanism
for arresting growth to reduce such risks was needed.
The 1800s also saw tremendous expansion in our scientific knowledge and
engineering capabilities with the consequent development of new techniques,
including the compression of gases to the liquid state, which enabled the field
of cryobiology to develop rapidly. The ability to use ultra-low temperatures to
prevent degradation of biological materials has probably been utilized by man
for millennia, and further scientific observations from the 17th and 18th cen-
turies paved the way for discoveries in cyropreservation technology in the
20th century. These led to successful and reliable methods for the preserva-
tion of both prokaryotic and eukaryotic organisms so they could be stored
indefinitely in a viable and unchanging state of “suspended animation.” The
preservation of bacteria and fungi had been established by pioneers from the
19th and early 20th centuries, subsequent work of people like Polge et al. (1)
for preservation of animal cells, and Sakai (2) for plant cells can be viewed as
key milestones in the development of cryopreservation processes. This pio-
neering work has been improved and refined with new approaches and funda-
mental research into cryobiology that has enabled the preservation of diverse
and complex cell and tissue cultures as exemplified in the protocols provided
in this book.
Today, culture collections, or more broadly, biological resource centers
(BRCs), are a mixture of academic, public service, private, government and com-
mercial activities that deliver important characterized cultures as “seed” stocks:
1. For the development of industrial processes.
2. As reference strains for biological assays and published scientific literature.
3. As type strains for taxonomical studies.
4. As centers for conservation of biodiversity.
In this chapter, we shall outline some of the important principles and chal-
lenges involved in the establishment and long-term maintenance of collections
of cryopreserved biological materials and cultures.

2. Fundamental Principles for BRCs

There are three fundamental features of collections of biological materials
that must be sustained to establish the value of stored material: (1) purity
(freedom from contaminant organisms); (2) authenticity (correct identity of
each strain), and (3) stability, including correct functional characteristics. Purity
of a strain is critical to avoid erroneous data. However, in some situations purity
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