Table Of ContentAdvances in
BIOPRESERVATION
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E di te d by
J O H N G . B A U S T
Institute of Biomedical Technology
State University of New York
Binghamton, New York
J O H N M . B A U S T
Cell Preservation Services, Inc.
Owego, New York
Boca Raton London New York
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Library of Congress Cataloging‑in‑Publication Data
Advances in biopreservation / edited by John G. Baust and John M. Baust.
p. cm.
Includes bibliographical references and index.
ISBN 0‑8493‑2772‑5
1. Cryobiology. I. Baust, J. G. II. Baust, John M.
QH324.9.C7A46 2006
571.4’645‑‑dc22
2006004020
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© 2007 by Taylor & Francis Group, LLC
Preface
Sixty-five years have passed since the publication of Basil Luyet’s
Life and Death at Low Temper-
ature
, the first organized attempt to summarize past observations of freezing injury in living
organisms and to infer from them the mechanisms of injury. Not surprisingly, attention at that time
was focused primarily on mechanical damage from ice, although, as evidence of the immaturity
of the science, Luyet considered five other possible mechanisms: the withdrawal of energy, attain-
ment of a minimal temperature, too-rapid thawing, dehydration, and “various physiological, phys-
ical, or chemical alterations.”
One might have expected that the subsequent years would have led to a clear and functional
understanding of freezing injury and, as a result, have enabled efficient cryopreservation of almost
anything. But it wasn’t that easy. Looking back at some of the hypotheses that emerged — salt
concentration (Lovelock), disulfide binding (Levitt), the two factor hypothesis (Mazur), minimum
volume (Meryman), membrane depletion (Williams, Steponkus) — one can see that, in retrospect,
each in a very general sense was correct, but none provided sufficient insight to lift applied
cryopreservation very far from the empirical. In fact, even after those sixty-plus years, our insight
is still pretty much limited to the recognition that intracellular ice is generally lethal, that ice nuclei
are rare or absent within cells, that extracellular ice concentrates the extracellular solution and the
cells lose water osmotically and shrink. However, the precise nature of the lethal event for each
cell type continues to be debatable, whether from the seeding of extracellular ice through membrane
pores, from membrane stresses leading to the loss of membrane material, from temperature- and
concentration-related denaturation or precipitation, or from some as-yet ill-defined phenomenon.
The role of cold denaturation of proteins has been largely ignored.
Fortunately — or, some might argue, unfortunately — the advent of penetrating cryoprotectants,
primarily glycerol and DMSO, has enabled empirical cryopreservation to leapfrog basic research.
Topics that were central to cryobiology research in the 1950s and 1960s have been overshadowed
by demonstrations of empirical success. Cryopreservation is increasingly being undertaken by
specialists in the subject tissue rather than by cryobiologists, and the practicalities of grant and
contract support tend to perpetuate this trend.
However, although the effectiveness of the penetrating cryoprotectants has made many basic
questions moot, the challenges are still far from resolved. The cryoprotectants themselves are now
the obstacles to easy success. Glycerol penetrates cells slowly and some cells not at all, creating
major osmotic problems. But glycerol has the great advantage of being a nontoxic macromolecular
stabilizer. DMSO, despite its reputation, does not cross membranes instantly; in fact, at temperatures
below 10°C, it enters cells relatively slowly — at least this is true for red cells. And it is a
macromolecular destabilizer with demonstrated toxicity.
From the purely practical aspect, it is better cryoprotectants that are needed. Polymers have
their own shortcomings and, with some exceptions, enable long-term, low-temperature storage only
in the presence of associated penetrating agents. Complete vitrification is a promising approach,
but there are still formidable obstacles to be surmounted. For an ideal penetrating agent, there are
only two criteria. Such an agent must cross cell membranes readily and rapidly and must be nontoxic
at elevated concentrations. Obvious candidates are low-molecular weight organic compounds or
cations such as ammonium compounds that are partially dissociated and enter the cell in the
uncharged form. However, there are a limited number of low-molecular weight compounds and
even the two simple criteria cited above may be too demanding.
2772_C000.fm Page v Tuesday, July 18, 2006 11:08 AM
© 2007 by Taylor & Francis Group, LLC
This timely and impressive volume shows just how far cryobiology has come in providing
frozen preservation for a variety of cells and tissues and also how much dedicated effort it has
taken to get this far. It is also apparent that each individual cell type requires an individually crafted
cryoprotective regimen that may still achieve only limited recovery, and that the ultimate goals of
applied cryobiology have yet to be achieved. It is likely that the limitations of cryopreservation
apparent today will not be resolved just by more tinkering, but will require a return to the basic
questions that drove cryobiology during its earlier years. In the pharmaceutical industry, examples
abound of successful new therapeutics made possible only by an accurate understanding of the
process to be addressed. For cryopreservation, the easy answers are not good enough. The time is
ripe for more basic science.
Harold T. Meryman
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© 2007 by Taylor & Francis Group, LLC
Editors
John G. Baust, Ph.D.,
holds an appointment as a lead professor in the Departments of Biological
Sciences and Bioengineering, and is the Director of the Institute of Biomedical Technology at the
State University of New York at Binghamton. Dr. Baust directs multidisciplinary research programs
in cell/tissue cryopreservation, hypothermic organ preservation, tissue engineering, and cancer
therapy. He serves on several advisory and editorial boards of various biotech corporations and
journals including serving as Editor-in-Chief for
Cell Preservation Technology
and board member
for the
International Society for Cryobiology
. He has authored and co-authored hundreds of papers,
reviews, and abstracts and holds over 50 U.S. and international patents in the area of the low-
temperature sciences. Dr. Baust has founded, co-founded, or served on the advisory board of
numerous companies, including Mariseed, Inc., LifeCell,, Inc., Cryomedical Sciences, Inc., and
Cryocath Technologies, Ltd. He presently serves as President and CEO of BioLife Solutions, Inc.
John M. Baust, Ph.D.,
serves as the President and CEO of Cell Preservation Services, Inc.
(CPSI) and is a research assistant professor in the Departments of Bioengineering and Biology at
the State University of New York at Binghamton. Prior to founding CPSI, Dr. Baust served as
Director of Research for BioLife Solutions, Inc., following completion of a postdoctoral research
fellowship at the Center for Engineering in Medicine at Harvard Medical School and Massachusetts
General Hospital. Dr. Baust, who received his Ph.D. in cell and molecular biology from the State
University of New York, directs multidisciplinary research programs in cell/tissue cryopreservation,
hypothermic organ preservation, tissue engineering and cancer therapy. He serves on several
advisory and editorial boards of various biotech corporations and journals including the journal
Cell Preservation Technology. He also serves on the Board of Governors of the Society for
Cryobiology. He has authored and co-authored numerous papers, reviews, abstracts, and patents in
the area of the low-temperature biology and has been instrumental in the advancement of the field
of cryobiology into the molecular biological era, focusing in the areas of signal transduction and
apoptosis.
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© 2007 by Taylor & Francis Group, LLC
Contributors
Jason P. Acker
Department of Laboratory Medicine &
Pathology
University of Alberta
Edmonton, AB
Joong-Hyuck Auh
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Rachna Bali
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Mitali Banerjee
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
John G. Baust
Institute of Biomedical Technology
SUNY Binghamton
Binghamton, NY
John M. Baust
Cell Preservation Services, Inc.
Owego, NY
Kelvin G. M. Brockbank
Cell and Tissue Systems, Inc.
Charleston, SC
Robert G. Van Buskirk
Department of Biological Sciences
SUNY Binghamton
Binghamton, NY
Dana Clutter
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
John H. Crowe
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Lois M. Crowe
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Denis Dwyre
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Erika Fisk
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Barry Fuller
University Department of Surgery
Royal Free & University College Medical
School
London, UK
Andrew A. Gage
Roswell Park Memorial Institute
SUNY Buffalo
Buffalo, NY
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© 2007 by Taylor & Francis Group, LLC
Dayong Gao
Department of Mechanical Engineering
University of Kentucky, College of Engineering
Lexington, KY
Karlis Gross
Department of Mechanical & Manufacturing
Engineering
University of Melbourne
Melbourne, Australia
Tariq Hafez
University Department of Surgery
Royal Free & University College Medical
School
London, UK
Allison Hubel
Department of Mechanical Engineering
University of Minnesota
Minneapolis, MN
Kamran Jamil
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Azadeh Kheirolomoom
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Chad Leidy
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Roger A. Leopold
USDA/ARS Biosciences Research Laboratory
Fargo, ND
Stephen Livesey
Chief Scientific Officer
Australian Stem Cell Center, Ltd.
Melbourne, Australia
Sheri Looper
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Dawei Luo
Department of Mechanical Engineering
University of Kentucky, College of Engineering
Lexington, KY
Xiaocui Ma
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Jeffrey Norris
Department of Cell Biology, Physiology, and
Anatomy
Center for Biostabilization
University of California
Davis, CA
Ann E. Oliver
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Yoed Rabin
Department of Mechanical Engineering
Carnegie Mellon University
Pittsburgh, PA
Resmi Ravindran
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Josette Ricker
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
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© 2007 by Taylor & Francis Group, LLC
Joe Russell
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Gyana Satpathy
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Kristi Snyder
Cell Preservation Services
Owego, NY
Paul S. Steif
Department of Mechanical Engineering
Carnegie Mellon University
Pittsburgh, PA
Fern Tablin
Department of Cell Biology, Physiology, and
Anatomy
Center for Biostabilization
University of California
Davis, CA
Minke Tang
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Michael J. Taylor
Cell and Tissue Systems, Inc.
Charleston, SC
Zsolt Torok
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Kathy Traianedes
Australian Stem Cell Center, Ltd.
Monash University,
Clayton, Australia
Nelly M. Tsvetkova
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Naomi Walker
Department of Cell Biology, Physiology, and
Anatomy
Center for Biostabilization
University of California
Davis, CA
Willem Wolkers
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
Jianping Yu
Department of Mechanical Engineering
University of Kentucky, College of Engineering
Lexington, KY
Shijun Zhu
Molecular and Cellular Biology
Center for Biostabilization
University of California
Davis, CA
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© 2007 by Taylor & Francis Group, LLC
Table of Contents
Chapter 1
Concepts in Biopreservation..............................................................................................................1
John G. Baust
Chapter 2
Biology of Cell Survival in the Cold: The Basis for Biopreservation of
Tissues and Organs ..........................................................................................................................15
Michael J. Taylor
Chapter 3
Properties of Cells and Tissues Influencing Preservation Outcome: Molecular
Basis of Preservation-Induced Cell Death.......................................................................................63
John M. Baust
Chapter 4
Selective Cryotherapy: Preservation-Ablation.................................................................................89
Andrew Gage, Kristi Snyder, and John M. Baust
Chapter 5
Biomaterials ...................................................................................................................................107
Kathy Traianedes, Karlis Gross,
and Stephen Livesey
Chapter 6
Viability and Functional Assays Used to Assess Preservation Efficacy:
The Multiple Endpoint/Tier Approach ..........................................................................................123
Robert G. Van Buskirk
Chapter 7
Preservation of Cellular Therapies: Gene Therapy, Cellular, and
Metabolic Engineering...................................................................................................................143
Allison Hubel
Chapter 8
Tissue Preservation ........................................................................................................................157
Kelvin G.M. Brockbank and Michael J. Taylor
Chapter 9
Applications: Organ Preservation for Transplantation..................................................................197
Tariq Hafez and Barry Fuller
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© 2007 by Taylor & Francis Group, LLC
Chapter 10
Cryopreservation of Nonmammalian Metazoan Systems.............................................................271
Roger A. Leopold
Chapter 11
The Use of Intracellular Protectants in Cell Biopreservation.......................................................299
Jason P. Acker
Chapter 12
Thermal Instruments and Devices in Cryobiological Research and Applications .......................321
Dayong Gao, Jianping Yu, and Dawei Luo
Chapter 13
Solid Mechanics Aspects of Cryobiology .....................................................................................359
Yoed Rabin and Paul S. Steif
Chapter 14
Stabilization of Mammalian Cells in the Dry State......................................................................383
John H. Crowe, Lois M. Crowe, Willem Wolkers, Nelly M. Tsvetkova,
Ann E. Oliver, Zsolt Torok, Azadeh Kheirolomoom, Jeffrey Norris, Gyana Satpathy,
Xiaocui Ma, Minke Tang, Joong-Hyuck Auh, Shijun Zhu, Mitali Banerjee,
Resmi Ravindran, Chad Leidy, Josette Ricker, Kamran Jamil, Naomi Walker,
Sheri Looper, Joe Russell, Erika Fisk, Rachna Bali, Dana Clutter,
Denis Dwyre, and Fern Tablin
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© 2007 by Taylor & Francis Group, LLC
